1 Running head: Sphingolipids in plant defense responses 2 ...€¦ · 16/9/2015 · 164 in...
Transcript of 1 Running head: Sphingolipids in plant defense responses 2 ...€¦ · 16/9/2015 · 164 in...
1
Running head Sphingolipids in plant defense responses 1
2
Corresponding author Sandrine Dhondt-Cordelier 3
Uniteacute de Recherche Vigne et Vin de Champagne 4
(URVVC-EA 4707) Laboratoire Stress Deacutefenses et 5
Reproduction des Plantes Universiteacute de Reims 6
Champagne-Ardenne BP 1039 F-51687 Reims 7
cedex 2 France 8
9
Telephone +33 326 918 587 10
Email sandrinecordelieruniv-reimsfr 11
12
13
Research area Signaling and Response 14
15
16
17
Plant Physiology Preview Published on September 16 2015 as DOI101104pp1501126
Copyright 2015 by the American Society of Plant Biologists
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
2
Modifications of sphingolipid content affect tolerance to 18
hemibiotrophic and necrotrophic pathogens by modulating 19
plant defense responses in Arabidopsis 20
21
Magnin-Robert Maryline1 Le Bourse Doriane2 Markham Jonathan2 Steacutephan 22
Dorey1 Cleacutement Christophe1 Baillieul Fabienne1 and Dhondt-Cordelier 23
Sandrine1 24
25 1 Uniteacute de Recherche Vigne et Vin de Champagne (URVVC-EA 4707) 26
Laboratoire Stress Deacutefenses et Reproduction des Plantes SFR Condorcet FR 27
CNRS 3417 Universiteacute de Reims Champagne-Ardenne BP 1039 F-51687 28
Reims cedex 2 France 29 2 Center for Plant Science Innovation and Department of Biochemistry 30
University of Nebraska-Lincoln Beadle Center 1901 Vine Street Lincoln NE 31
68588 USA 32
33
34
One-sentence summary 35
Sphingolipids play a key role in plant defense towards different lifestyle 36
pathogens by modulating cell death ROS accumulation and jasmonate 37
signaling pathway 38 39
40
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
Footnotes 41 42 This work was supported in part by a grant (EliDeRham project ndash A2101-03) 43 from the Region Champagne-Ardenne 44
45 46 Corresponding author Sandrine Dhondt-Cordelier 47
Email sandrinecordelieruniv-reimsfr 48 49 50
51
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 52
53
Sphingolipids are emerging as second messengers in programmed cell death 54
and plant defense mechanisms However their role in plant defense is far from 55
being understood especially against necrotrophic pathogens 56
Sphingolipidomics and plant defense responses during pathogenic infection 57
were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase 58
encoded by the AtDPL1 gene and regulating LCBLCB-P homeostasis Atdpl1 59
mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but 60
susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv 61
tomato (Pst) Here a direct comparison of sphingolipid profiles during infection 62
with pathogen differing in lifestyles is described In contrast to LCBs (d180 and 63
d182) hydroxyceramide and LCB-P (t180-P and t181-P) levels are higher in 64
Atdpl1-1 than in WT plants in response to B cinerea Following Pst infection 65
t180-P accumulates more strongly in Atdpl1-1 than in WT plants Moreover 66
d180 and t180-P appears as key players in Pst- and B cinerea-induced cell 67
death and reactive oxygen species accumulation Salicylic acid (SA) levels are 68
similar in both types of plants independently of the pathogen In addition SA-69
dependent gene expression is similar in both types of B cinerea-infected plants 70
but is repressed in Atdpl1-1 after treatment with Pst Both pathogen infection 71
triggers higher jasmonic acid (JA) JA-Ile accumulation and JA-dependent gene 72
expression in Atdpl1-1 mutants Our results demonstrate that sphingolipids play 73
an important role in plant defense especially towards necrotrophic pathogen 74
and highlight a novel connection between jasmonate signaling pathway cell 75
death and sphingolipids 76
77 78
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 79
80
Plants have evolved a complex array of defenses when attacked by 81
microbial pathogens The success of plant resistance firstly relies on the 82
capacity of the plant to recognize its invader Among early events a transient 83
production of reactive oxygen species (ROS) known as oxidative burst is 84
characteristic of successful pathogen recognition (Torres 2010) Perception of 85
pathogen attack then initiates a large array of immune responses including 86
modification of cell walls as well as the production of anti-microbial proteins and 87
metabolites like pathogenesis-related (PR) proteins and phytoalexins 88
respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89
acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90
networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91
Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92
signal molecules allow the plant to activate andor modulate an appropriate 93
array of defense responses depending on the pathogen lifestyle necrotroph or 94
biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95
considered as essential for resistance to (hemi)biotrophic pathogens it is 96
assumed that JA and ET signaling pathways are important for resistance to 97
necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98
2005) A successful innate immune response often includes the so-called 99
hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100
occurring in a limited area at the site of infection This suicide of infected cells is 101
thought to limit the spread of biotrophic pathogens including viruses bacteria 102
fungi and oomycetes (Mur et al 2008) 103
During the past decade significant progress has been made in our 104
understanding of the cellular function of plant sphingolipids Besides being 105
structural components of cell membranes sphingolipids are bioactive 106
metabolites that regulate important cellular processes such as cell survival and 107
PCD occurring during either plant development or plant defense (Dunn et al 108
2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109
sphingolipids in these processes came from the use of the fungal toxins 110
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
2
Modifications of sphingolipid content affect tolerance to 18
hemibiotrophic and necrotrophic pathogens by modulating 19
plant defense responses in Arabidopsis 20
21
Magnin-Robert Maryline1 Le Bourse Doriane2 Markham Jonathan2 Steacutephan 22
Dorey1 Cleacutement Christophe1 Baillieul Fabienne1 and Dhondt-Cordelier 23
Sandrine1 24
25 1 Uniteacute de Recherche Vigne et Vin de Champagne (URVVC-EA 4707) 26
Laboratoire Stress Deacutefenses et Reproduction des Plantes SFR Condorcet FR 27
CNRS 3417 Universiteacute de Reims Champagne-Ardenne BP 1039 F-51687 28
Reims cedex 2 France 29 2 Center for Plant Science Innovation and Department of Biochemistry 30
University of Nebraska-Lincoln Beadle Center 1901 Vine Street Lincoln NE 31
68588 USA 32
33
34
One-sentence summary 35
Sphingolipids play a key role in plant defense towards different lifestyle 36
pathogens by modulating cell death ROS accumulation and jasmonate 37
signaling pathway 38 39
40
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
Footnotes 41 42 This work was supported in part by a grant (EliDeRham project ndash A2101-03) 43 from the Region Champagne-Ardenne 44
45 46 Corresponding author Sandrine Dhondt-Cordelier 47
Email sandrinecordelieruniv-reimsfr 48 49 50
51
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 52
53
Sphingolipids are emerging as second messengers in programmed cell death 54
and plant defense mechanisms However their role in plant defense is far from 55
being understood especially against necrotrophic pathogens 56
Sphingolipidomics and plant defense responses during pathogenic infection 57
were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase 58
encoded by the AtDPL1 gene and regulating LCBLCB-P homeostasis Atdpl1 59
mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but 60
susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv 61
tomato (Pst) Here a direct comparison of sphingolipid profiles during infection 62
with pathogen differing in lifestyles is described In contrast to LCBs (d180 and 63
d182) hydroxyceramide and LCB-P (t180-P and t181-P) levels are higher in 64
Atdpl1-1 than in WT plants in response to B cinerea Following Pst infection 65
t180-P accumulates more strongly in Atdpl1-1 than in WT plants Moreover 66
d180 and t180-P appears as key players in Pst- and B cinerea-induced cell 67
death and reactive oxygen species accumulation Salicylic acid (SA) levels are 68
similar in both types of plants independently of the pathogen In addition SA-69
dependent gene expression is similar in both types of B cinerea-infected plants 70
but is repressed in Atdpl1-1 after treatment with Pst Both pathogen infection 71
triggers higher jasmonic acid (JA) JA-Ile accumulation and JA-dependent gene 72
expression in Atdpl1-1 mutants Our results demonstrate that sphingolipids play 73
an important role in plant defense especially towards necrotrophic pathogen 74
and highlight a novel connection between jasmonate signaling pathway cell 75
death and sphingolipids 76
77 78
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 79
80
Plants have evolved a complex array of defenses when attacked by 81
microbial pathogens The success of plant resistance firstly relies on the 82
capacity of the plant to recognize its invader Among early events a transient 83
production of reactive oxygen species (ROS) known as oxidative burst is 84
characteristic of successful pathogen recognition (Torres 2010) Perception of 85
pathogen attack then initiates a large array of immune responses including 86
modification of cell walls as well as the production of anti-microbial proteins and 87
metabolites like pathogenesis-related (PR) proteins and phytoalexins 88
respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89
acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90
networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91
Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92
signal molecules allow the plant to activate andor modulate an appropriate 93
array of defense responses depending on the pathogen lifestyle necrotroph or 94
biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95
considered as essential for resistance to (hemi)biotrophic pathogens it is 96
assumed that JA and ET signaling pathways are important for resistance to 97
necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98
2005) A successful innate immune response often includes the so-called 99
hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100
occurring in a limited area at the site of infection This suicide of infected cells is 101
thought to limit the spread of biotrophic pathogens including viruses bacteria 102
fungi and oomycetes (Mur et al 2008) 103
During the past decade significant progress has been made in our 104
understanding of the cellular function of plant sphingolipids Besides being 105
structural components of cell membranes sphingolipids are bioactive 106
metabolites that regulate important cellular processes such as cell survival and 107
PCD occurring during either plant development or plant defense (Dunn et al 108
2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109
sphingolipids in these processes came from the use of the fungal toxins 110
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
3
Footnotes 41 42 This work was supported in part by a grant (EliDeRham project ndash A2101-03) 43 from the Region Champagne-Ardenne 44
45 46 Corresponding author Sandrine Dhondt-Cordelier 47
Email sandrinecordelieruniv-reimsfr 48 49 50
51
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 52
53
Sphingolipids are emerging as second messengers in programmed cell death 54
and plant defense mechanisms However their role in plant defense is far from 55
being understood especially against necrotrophic pathogens 56
Sphingolipidomics and plant defense responses during pathogenic infection 57
were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase 58
encoded by the AtDPL1 gene and regulating LCBLCB-P homeostasis Atdpl1 59
mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but 60
susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv 61
tomato (Pst) Here a direct comparison of sphingolipid profiles during infection 62
with pathogen differing in lifestyles is described In contrast to LCBs (d180 and 63
d182) hydroxyceramide and LCB-P (t180-P and t181-P) levels are higher in 64
Atdpl1-1 than in WT plants in response to B cinerea Following Pst infection 65
t180-P accumulates more strongly in Atdpl1-1 than in WT plants Moreover 66
d180 and t180-P appears as key players in Pst- and B cinerea-induced cell 67
death and reactive oxygen species accumulation Salicylic acid (SA) levels are 68
similar in both types of plants independently of the pathogen In addition SA-69
dependent gene expression is similar in both types of B cinerea-infected plants 70
but is repressed in Atdpl1-1 after treatment with Pst Both pathogen infection 71
triggers higher jasmonic acid (JA) JA-Ile accumulation and JA-dependent gene 72
expression in Atdpl1-1 mutants Our results demonstrate that sphingolipids play 73
an important role in plant defense especially towards necrotrophic pathogen 74
and highlight a novel connection between jasmonate signaling pathway cell 75
death and sphingolipids 76
77 78
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 79
80
Plants have evolved a complex array of defenses when attacked by 81
microbial pathogens The success of plant resistance firstly relies on the 82
capacity of the plant to recognize its invader Among early events a transient 83
production of reactive oxygen species (ROS) known as oxidative burst is 84
characteristic of successful pathogen recognition (Torres 2010) Perception of 85
pathogen attack then initiates a large array of immune responses including 86
modification of cell walls as well as the production of anti-microbial proteins and 87
metabolites like pathogenesis-related (PR) proteins and phytoalexins 88
respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89
acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90
networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91
Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92
signal molecules allow the plant to activate andor modulate an appropriate 93
array of defense responses depending on the pathogen lifestyle necrotroph or 94
biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95
considered as essential for resistance to (hemi)biotrophic pathogens it is 96
assumed that JA and ET signaling pathways are important for resistance to 97
necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98
2005) A successful innate immune response often includes the so-called 99
hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100
occurring in a limited area at the site of infection This suicide of infected cells is 101
thought to limit the spread of biotrophic pathogens including viruses bacteria 102
fungi and oomycetes (Mur et al 2008) 103
During the past decade significant progress has been made in our 104
understanding of the cellular function of plant sphingolipids Besides being 105
structural components of cell membranes sphingolipids are bioactive 106
metabolites that regulate important cellular processes such as cell survival and 107
PCD occurring during either plant development or plant defense (Dunn et al 108
2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109
sphingolipids in these processes came from the use of the fungal toxins 110
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
4
ABSTRACT 52
53
Sphingolipids are emerging as second messengers in programmed cell death 54
and plant defense mechanisms However their role in plant defense is far from 55
being understood especially against necrotrophic pathogens 56
Sphingolipidomics and plant defense responses during pathogenic infection 57
were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase 58
encoded by the AtDPL1 gene and regulating LCBLCB-P homeostasis Atdpl1 59
mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but 60
susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv 61
tomato (Pst) Here a direct comparison of sphingolipid profiles during infection 62
with pathogen differing in lifestyles is described In contrast to LCBs (d180 and 63
d182) hydroxyceramide and LCB-P (t180-P and t181-P) levels are higher in 64
Atdpl1-1 than in WT plants in response to B cinerea Following Pst infection 65
t180-P accumulates more strongly in Atdpl1-1 than in WT plants Moreover 66
d180 and t180-P appears as key players in Pst- and B cinerea-induced cell 67
death and reactive oxygen species accumulation Salicylic acid (SA) levels are 68
similar in both types of plants independently of the pathogen In addition SA-69
dependent gene expression is similar in both types of B cinerea-infected plants 70
but is repressed in Atdpl1-1 after treatment with Pst Both pathogen infection 71
triggers higher jasmonic acid (JA) JA-Ile accumulation and JA-dependent gene 72
expression in Atdpl1-1 mutants Our results demonstrate that sphingolipids play 73
an important role in plant defense especially towards necrotrophic pathogen 74
and highlight a novel connection between jasmonate signaling pathway cell 75
death and sphingolipids 76
77 78
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 79
80
Plants have evolved a complex array of defenses when attacked by 81
microbial pathogens The success of plant resistance firstly relies on the 82
capacity of the plant to recognize its invader Among early events a transient 83
production of reactive oxygen species (ROS) known as oxidative burst is 84
characteristic of successful pathogen recognition (Torres 2010) Perception of 85
pathogen attack then initiates a large array of immune responses including 86
modification of cell walls as well as the production of anti-microbial proteins and 87
metabolites like pathogenesis-related (PR) proteins and phytoalexins 88
respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89
acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90
networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91
Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92
signal molecules allow the plant to activate andor modulate an appropriate 93
array of defense responses depending on the pathogen lifestyle necrotroph or 94
biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95
considered as essential for resistance to (hemi)biotrophic pathogens it is 96
assumed that JA and ET signaling pathways are important for resistance to 97
necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98
2005) A successful innate immune response often includes the so-called 99
hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100
occurring in a limited area at the site of infection This suicide of infected cells is 101
thought to limit the spread of biotrophic pathogens including viruses bacteria 102
fungi and oomycetes (Mur et al 2008) 103
During the past decade significant progress has been made in our 104
understanding of the cellular function of plant sphingolipids Besides being 105
structural components of cell membranes sphingolipids are bioactive 106
metabolites that regulate important cellular processes such as cell survival and 107
PCD occurring during either plant development or plant defense (Dunn et al 108
2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109
sphingolipids in these processes came from the use of the fungal toxins 110
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
5
INTRODUCTION 79
80
Plants have evolved a complex array of defenses when attacked by 81
microbial pathogens The success of plant resistance firstly relies on the 82
capacity of the plant to recognize its invader Among early events a transient 83
production of reactive oxygen species (ROS) known as oxidative burst is 84
characteristic of successful pathogen recognition (Torres 2010) Perception of 85
pathogen attack then initiates a large array of immune responses including 86
modification of cell walls as well as the production of anti-microbial proteins and 87
metabolites like pathogenesis-related (PR) proteins and phytoalexins 88
respectively (Schwessinger and Ronald 2012) The plant hormones salicylic 89
acid (SA) jasmonic acid (JA) and ethylene (ET) are key players in the signaling 90
networks involved in plant resistance (Bari and Jones 2009 Tsuda and 91
Katagiri 2010 Robert-Seilaniantz et al 2011) Interactions between these 92
signal molecules allow the plant to activate andor modulate an appropriate 93
array of defense responses depending on the pathogen lifestyle necrotroph or 94
biotroph (Glazebrook 2005 Koornneef and Pieterse 2008) Whereas SA is 95
considered as essential for resistance to (hemi)biotrophic pathogens it is 96
assumed that JA and ET signaling pathways are important for resistance to 97
necrotrophic pathogens in Arabidopsis (Thomma et al 2001 Glazebrook 98
2005) A successful innate immune response often includes the so-called 99
hypersensitive response (HR) a form of rapid programmed cell death (PCD) 100
occurring in a limited area at the site of infection This suicide of infected cells is 101
thought to limit the spread of biotrophic pathogens including viruses bacteria 102
fungi and oomycetes (Mur et al 2008) 103
During the past decade significant progress has been made in our 104
understanding of the cellular function of plant sphingolipids Besides being 105
structural components of cell membranes sphingolipids are bioactive 106
metabolites that regulate important cellular processes such as cell survival and 107
PCD occurring during either plant development or plant defense (Dunn et al 108
2004 Berkey et al 2012 Markham et al 2013) First evidence of the role of 109
sphingolipids in these processes came from the use of the fungal toxins 110
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
6
fumonisin B1 (FB1) and AAL produced by the necrotrophic agent Alternaria 111
alternata f sp lycopersici These toxins are structural sphingosine (d181) 112
analogs and function as ceramide synthase inhibitors They triggered PCD 113
when exogenously applied to plants Mutant strains in which production of such 114
toxin is abrogated failed to infect the host plant implying that toxin 115
accumulation is required for pathogenicity and that induction of plant PCD could 116
be considered as a virulence tool used by necrotrophic pathogen (Berkey et al 117
2012) Moreover several studies revealed that ceramides (Cers) and long-chain 118
bases (LCBs) are also potent inducers of PCD in plants For example 119
exogenously applied Cers and LCBs (d180 d181 or t180) induced PCD either 120
in cell suspension cultures (Liang et al 2003 Lachaud et al 2010 Alden et 121
al 2011 Lachaud et al 2011) or in whole seedlings (Shi et al 2007 122
Takahashi et al 2009 Saucedo-Garcia et al 2011) AAL- and FB1-induced 123
PCD seemed to be due to the accumulation of free sphingoid bases (d180 and 124
t180) (Abbas et al 1994 Brandwagt et al 2000 Shi et al 2007) 125
Spontaneous cell death in lag one homolog 1 (loh1) or L-myo-inositol 1-126
phosphate synthase (mips) mutant could be due to trihydroxy-LCB andor Cer 127
accumulation (Donahue et al 2010 Ternes et al 2011) Deciphering of Cer 128
participation in induction of HR and associated PCD also came from studies on 129
accelerated cell death 5 (acd5) and enhancing RPW8-mediated HR (erh1) 130
mutants which displayed over-accumulation of Cers These mutants exhibited 131
spontaneous cell death and resistance to biotrophic pathogen which seemed to 132
be linked with SA and PR-protein accumulation (Liang et al 2003 Wang et al 133
2008) Altogether these data provide evidence of a link between PCD defense 134
and sphingolipid metabolism However the fatty acid hydroxylase 12 135
(atfah1atfah2) double mutant that accumulates SA and Cers was more tolerant 136
to the obligate biotrophic fungus Golovinomyces cichoracearum but did not 137
display a PCD-like phenotype suggesting that Cers alone are not involved in 138
the induction of PCD (Koumlnig et al 2012) Moreover Saucedo-Garcia et al 139
(2011) postulated that dihydroxy-LCBs but not trihydroxy-LCBs might be 140
primary mediators for LCB-induced PCD The LCB C-4 hydroxylase sbh1sbh2 141
double mutant completely lacking trihydroxy-LCBs showed enhanced 142
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
7
expression of PCD marker genes (Chen et al 2008) On the contrary increase 143
in t180 was specifically sustained in plant interaction with the avirulent Pst 144
strain and correlated with a strong PCD induction in leaves (Peer et al 2010) 145
Thus the nature of sphingolipids able to induce PCD is still under debate and 146
may evolve depending on plants and their environment Phosphorylated form of 147
LCBs (LCB-Ps) could abrogate PCD induced by LCBs Cers or heat stress in a 148
dose-dependent manner (Shi et al 2007 Alden et al 2011) Furthermore 149
blocking conversion of LCBs to LCB-Ps by using specific inhibitors induced 150
PCD in cell suspension culture (Alden et al 2011) Recently overexpression of 151
rice LCB kinase in transgenic tobacco plants reduced PCD after treatment with 152
FB1 (Zhang et al 2013) Genetic mutation on LCB-P lyase encoded by the 153
AtDPL1 gene modifying LCBLCB-P ratio could impact PCD levels after 154
treatment with FB1 (Tsegaye et al 2007) Altogether these data point to the 155
existence of a rheostat between LCBs and their phosphorylated forms that 156
controls plant cell fate toward cell death or survival 157
Data on plant sphingolipid functions are still fragmentary Only few reports 158
described interconnections between sphingolipids cell death and plant defense 159
responses almost exclusively in response to (hemi)biotrophic pathogens 160
Knowledge about such relation in response to necrotrophic pathogen is still in 161
its infancy (Rivas-San Vicente et al 2013 Bi et al 2014) In the present report 162
the link between sphingolipids cell death and plant defense has been explored 163
in response to B cinerea infection and in comparison to Pst infection For this 164
purpose Atdpl1 mutant plants disturbed in LCBLCB-P accumulation without 165
displaying any phenotype under standard growth conditions (Tsegaye et al 166
2007) have been analyzed after pathogen infection Our results revealed that 167
modification of sphingolipid contents not only impacted plant tolerance to 168
hemibiotrophs but also greatly affected resistance to necrotrophs Whereas SA 169
signaling pathway is globaly repressed in Atdpl1-1 compared to wild type (WT) 170
plants JA signaling pathway is significantly enhanced Cell death and ROS 171
accumulation are markedly modified in Atdpl1-1 mutant plants We further 172
demonstrated that t180-P and d180 are key players in pathogen-induced cell 173
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
8
death and ROS generation Here we thus established a link between JA 174
signaling PCD and sphingolipid metabolism 175
176
177
RESULTS 178
Necrotrophic and hemibiotrophic infection differently affect Atdpl1 mutant 179
plant response 180
In order to assess the role of sphingolipids in the plant immune responses to 181
necrotrophic and hemibiotrophic pathogens we used Atdpl1 mutant which is 182
affected in the LCBLCB-P rheostat by accumulating t181-P (Tsegaye et al 183
2007) Whereas Atdpl1 mutant shows no developmental phenotype compared 184
to WT plants under standard conditions it exhibits a higher sensitivity to FB1 185
(Tsegaye et al 2007) B cinerea or Pst have been widely used to decipher 186
defense mechanisms to necrotrophic and hemibiotrophic pathogens in 187
Arabidopsis (Glazebrook 2005) To get some information about the 188
susceptibility of Atdpl1 mutant to B cinerea or Pst (either virulent (Pst DC3000) 189
or avirulent (Pst AvrRPM1) strain) three independent Atdpl1 mutant lines were 190
thus challenged with these pathogens The three Atdpl1 mutant lines displayed 191
similar responses upon pathogen challenge (Fig 1) In B cinerea-infected WT 192
plants disease symptoms showing chlorosis and necrosis increased more 193
rapidly than in B cinerea-infected Atdpl1 plants (Fig 1A) On the contrary 194
symptoms developed in response to Pst infection were more pronounced in 195
mutant plants than in WT plants (Fig 1A) The lesion diameters were scored 48 196
and 60 h after drop-inoculation with B cinerea and classified in size categories 197
(Fig 1B) Interestingly Atdpl1 plants did not display necrotic lesion of the 198
largest size whereas WT plants showed 10 of these lesions 48 hpi Only 2 199
of the largest lesions were observed in Atdpl1 plants compared to 12 for WT 200
plants 60 hpi respectively Furthermore Atdpl1 mutants displayed a greater 201
percentage of small necrotic lesions than WT plants Atdpl1 lines displayed 202
approximately 45 and 65 of small lesions whereas WT showed only 17 203
and 24 of small lesions 48 and 60 hpi respectively Consequently fewer 204
lesions of medium size were observed in Atdpl1 lines than in WT plants (Fig 205
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
9
1B) The average of lesion diameters in Atdpl1 mutant was significantly lower 206
than in WT plants (plt001 plt0005) (Fig 1B) Plants were also infiltrated 207
with Pst DC3000 or Pst AvrRPM1 at 107 cfu mL-1 and bacterial populations 208
were evaluated 0 6 24 30 48 and 54 hpi As already described avirulent 209
strain growth was less important compared to virulent strain in WT plants (Fig 210
1 C and D) Interestingly infection with both bacterial strains revealed an 211
increased susceptibility of Atdpl1 plants allowing about tenfold more bacterial 212
growth as compared with WT plants (Fig 1 C and D) These results were also 213
correlated by fungal and bacterial population quantification in infected leaves by 214
qRT-PCR (Fig 1E) Interestingly AtDPL1 expression profile was similar after 215
either B cinerea or Pst infection (Supplemental Fig S1) Until 12 hpi no 216
AtDPL1 transcript accumulation could be observed AtDPL1 expression 217
significantly increased between 12 and 24 hpi and continuously rose until the 218
later stages of infection Symptoms due to either B cinerea invasion as well as 219
infection with virulent or avirulent strain of Pst visually appeared between 24 220
and 30 hpi (data not shown) thus are slightly delayed compared to AtDPL1 221
expression Deregulation of photosynthesis is considered as a tool for 222
evaluating the first sign of pathogen infection (Berger et al 2007 Bolton 223
2009) Repression of the RbcS gene (encoding the small subunit of ribulose-224
15-bisphosphate carboxylase) after pathogen infection occurred at the same 225
time (B cinerea) or slightly earlier (Pst) compared to AtDPL1 expression and 226
symptom appearance (Supplemental Fig S1) suggesting that an immediate 227
consequence of pathogen perception includes induction of AtDPL1 gene 228
expression Collectively these data indicate that lack of AtDPL1 activity in 229
mutant plants significantly delays the development of lesions triggered by B 230
cinerea infection but renders plants more susceptible to Pst infection 231
232
Sphingolipid profiles in WT and Atdpl1-1 plants are affected but differently 233
upon pathogen infection 234
To determine whether changes in the level of certain sphingolipids are 235
responsible for the delayed development of B cinerea infection in Atdpl1 236
mutant sphingolipid profiles were analyzed The main sphingolipid species in 237
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
10
Arabidopsis LCBs and LCB-Ps (Fig 2) glycosylinositolphosphoceramides 238
(GIPCs) (Fig 3) Cers (Fig 4) hydroxyceramides (hCers) (Fig 5) and 239
glucosylceramides (GlcCers) (Supplemental Fig S2) were first quantified in 240
both WT and Atdpl1-1 mutant at 0 hpi (Supplemental Fig S3) In WT and 241
Atdpl1-1 mutant plants LCBLCB-P basal levels were almost in the same range 242
than those already described in Tsegaye et al (2007) (Supplemental Fig S3) 243
As previously described the only significant alteration in sphingolipid basal 244
levels observed in Atdpl1-1 mutant compared to WT under typical growth 245
conditions was an increase in one specific LCB-P (t181-P) (Tsegaye et al 246
2007) (Supplemental Fig S3) Then we investigated the influence of B cinerea 247
infection on the sphingolipid profile in WT plants B cinerea infection triggered 248
LCB accumulation (from x6 for d182 to x20 for d180) (Fig 2A) but also a 249
moderate increase in d181-P and t181-P amount (x4 and x25 respectively) 250
compared to mock-inoculated WT plants (Fig 2E) The amount of total GIPCs 251
and more precisely saturated α-hydroxylated VLCFA-containing GIPCs (C24 252
and C26) (Fig 3 A and C) was significantly lower after B cinerea infection 253
than mock-treated plants (200 nmol g-1 DW and 300 nmol g-1 DW respectively) 254
(Supplemental Fig S4) Moreover d180- d181- and t181-GIPCs levels were 255
also reduced after B cinerea infection (Fig 3 A and C) Amount of total Cers is 256
4 times higher in B cinerea- than in mock-inoculated WT plants (84 vs 21 nmol 257
g-1 DW) (Supplemental Fig S4) Most of Cer molecules were affected by the 258
presence of B cinerea (Fig 4 A and C) Finally level of total hCers was not 259
modified (Supplemental Fig S4) however significant accumulation of saturated 260
α-hydroxylated C16- C18- and C26-containing hCers and d180-hCer was 261
observed after challenge with B cinerea (Fig 5 A and C) No change could be 262
noticed in GlcCer levels (Supplemental Fig S2 Supplemental Fig S4) To 263
better understand the role of sphingolipids in plant resistance to the 264
necrotrophic fungus a comparison between sphingolipid profiles in B cinerea-265
infected Atdpl1-1 mutant and WT plants was then performed With respect to 266
the LCBLCB-P pool WT plants contained more LCBs (Supplemental Fig S4) 267
especially d180 and d182 (Fig 2 A and B) whereas Atdpl1-1 mutant 268
accumulated more LCB-Ps (Supplemental Fig S4) especially t180-P and 269
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
11
t181-P (9 and 18-fold respectively) when compared to WT plants (Fig 2 E 270
and F) The amount of total GIPCs and more precisely saturated α-271
hydroxylated VLCFA-containing GIPCs (C22 C24 and C26) (Fig 3 C and D) 272
was significantly higher in Atdpl1-1 mutant than WT-treated plants after B 273
cinerea infection (370 vs 220 nmol g-1 DW respectively) (Supplemental Fig 274
S4) Total Cer amount was similar in both types of plants (Fig 4 C and D 275
Supplemental Fig S4) but B cinerea infection triggered an increased in hCer 276
contents especially saturated and mono-unsaturated VLCFA-containing hCers 277
(Fig 5 C and D) in Atdpl1-1 mutant compared to WT plants (75 vs 27 nmol g-1 278
DW respectively) (Supplemental Fig S4) Moreover trihydroxy-hCers also 279
accumulated three times in the mutant compared to WT plants in response to 280
the fungus (Fig 5 C and D) No significant change was observed in total 281
GlcCer amount (Supplemental Fig S2 Supplemental Fig S4) 282
In order to compare sphingolipid profile in response to an hemibiotrophic 283
pathogen analyses were performed 48 h after WT plant inoculation with 284
avirulent or virulent strains of Pst Our data confirmed previous results showing 285
that sphingolipid increase was more sustained during the incompatible than 286
compatible interaction (Peer et al 2010) Increase in t180 was observed in 287
response to both types of bacteria but infection with only Pst AvrRPM1 288
triggered a significant decrease of d181 (Fig 2C) After infection with Pst 289
AvrRPM1 an increase in d182-P t180-P and t181-P was observed whereas 290
only t180-P level was increased in response to Pst DC3000 (Fig 2G) GIPC 291
levels were also not significantly modified in response to both types of bacteria 292
(Fig3 E G and I Supplemental Fig S4) Total contents of d180- d181- 293
t180- and t181-Cers were increased after infection with Pst AvrRPM1 (Fig 4 294
E and I) Only an increase in trihydroxy-Cers could be noticed in response to 295
Pst DC3000 (Fig 4 E and G) Moreover t180-Cer level was higher in the case 296
of the incompatible interaction than in the case of the compatible one (40 vs 24 297
nmol g-1 DW respectively) (Supplemental Fig S4) C16- C24- and C26-Cers 298
also accumulated in response to both strains of Pst (Fig 4 E G and I) and 299
only C16-Cer accumulation was more pronounced in the case of interaction with 300
Pst AvrRPM1 compared to Pst DC3000 (45 vs 18 nmol g-1 DW respectively) 301
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
12
(Fig 4 E G and I) Total contents of d180-hCers were increased in response 302
to Pst (Fig 5 E G and I) t180-hCers accumulated after challenge with virulent 303
strain and t181-hCers after challenge with avirulent strain (Fig 5 E G and I) 304
Similarly to B cinerea infection no regulation of GclCer content could be 305
noticed (Supplemental Fig S2 Supplemental Fig S4) Comparison of 306
sphingolipid profiles between Pst-infected WT and Atdpl1-1 mutant plants 307
revealed an increase in d180 (x15) in Atdpl1-1 plants certainly due to 308
infiltration since it was also observed in control plants An increase in t180-P 309
level (x5) was however detected in Atdpl1-1 mutant plants compared to WT only 310
in response to the avirulent strain (Fig 2H) No significant regulation of GIPC 311
Cer hCer or GclCer pools was observed in response to either virulent or 312
avirulent strain (Fig 3 4 5 and Supplemental Fig S2) 313
314
Changes in sphingolipid profiles affect pathogen-induced cell death 315
Recently several reports have revealed that some sphingolipids are important 316
players in HR and associated PCD (Berkey et al 2012 Markham et al 2013) 317
HR is an effective strategy of plants to protect themselves against 318
(hemi)biotrophic microorganisms (Coll et al 2011) In contrast PCD processes 319
promote the spread of necrotrophic pathogens such as B cinerea (Govrin and 320
Levine 2000 Govrin et al 2006) Thus changes in sphingolipid profiles and 321
differences in tolerance upon B cinerea or Pst infection prompted us to 322
examine cell death response upon pathogen attack We thus measured 323
electrolyte leakage to detect changes in loss of ions caused by plasma 324
membrane damage characteristic of plant cell death (Dellagi et al 1998 325
Kawasaki et al 2005) Ion leakage measured after inoculation of Atdpl1-1 326
plants with B cinerea or Pst was reduced compared to WT plants (Fig 6 A and 327
B) These results suggested that modification in sphingolipid content could play 328
a role in modulating cell death processes in response to pathogen infection 329
Expression levels of PCD marker genes such as flavin-containing 330
monooxygenase FMO and senescence-associated genes SAG12 and SAG13 331
(Brodersen et al 2002) were also evaluated in order to verify if cell death 332
responses are modified in Atdpl1-1 mutant plants (Fig 7) FMO and SAG13 333
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
13
were induced in both types of plants with increasing infection spread of B 334
cinerea Interestingly these inductions occurred earlier and stronger in WT 335
(between 12 and 24 hpi) than in Atdpl1-1 mutant (between 24 and 30 hpi) (Fig 336
7 A and C) SAG12 was only induced 48 hpi in both WT and Atdpl1-1 mutant 337
and similarly to SAG13 and FMO its expression was stronger in WT (x 10000) 338
than in Atdpl1-1 mutant (x 2000) (Fig 7E) 339
As expected in the case of Pst infection SAG13 and FMO gene expressions 340
were induced earlier and stronger during the incompatible interaction than 341
during the compatible interaction (Fig 7 B and D) WT and mutant plants 342
displayed similar expression profiles with both types of bacteria however 343
induction was less pronounced in Atdpl1-1 mutant plants Similarly to B cinerea 344
infection SAG12 transcript accumulation occurred only at the later stages of the 345
infection (Fig 7F) It is noteworthy that induction of these PCD marker genes 346
followed a similar pattern than AtDPL1 gene expression in WT plants in 347
response to either B cinerea or Pst infection (Supplemental Fig S1 A and B) 348
SAG12 is only expressed in senescent tissues In contrast SAG13 and FMO 349
are expressed in different PCD processes (Lohman et al 1994 Brodersen et 350
al 2002) Collectively our data suggest that the induction of SAG13 and FMO 351
after either B cinerea or Pst infection could result from a HR-like PCD whereas 352
a senescence program is activated later This could also explain the tolerance 353
of Atdpl1 mutant plants towards B cinerea and their higher susceptibility 354
towards Pst 355
356
Modification of sphingolipid contents affect ROS production in response 357
to pathogen infection 358
Transient production of ROS is a hallmark of successful pathogen recognition 359
(Torres 2010) To investigate whether sphingolipid content perturbation in 360
Atdpl1-1 plants affected pathogen recognition we compared ROS production in 361
the mutant versus WT plants WT plants displayed a transient oxidative burst 362
peaking around 300 (B cinerea) or 40 (Pst) min after inoculation with B cinerea 363
or Pst respectively (Fig 8) This transient burst was significantly induced by 25 364
times in B cinerea-infected Atdpl1-1 plants compared to WT plants (Fig 8A) 365
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-
14
On the contrary ROS levels were significantly reduced in Pst-infected Atdpl1-1 366
plants compared to Pst-infected WT plants (Fig 8 B and C) Our results thus 367
demonstrated that signaling events linked to pathogen recognition are affected 368
by sphingolipid perturbation in Atdpl1-1 plants 369
370
Exogenous t180-P and d180 differently modifies pathogen-induced cell 371
death and ROS production 372
Major changes in LCB-P contents in B cinerea-inoculated Atdpl1-1 mutant 373
plants is an increase in t180-P levels and a decrease in d180 amounts (Fig 2) 374
We thus tested the ability of these sphingolipids to modulate pathogen-induced 375
cell death (Fig 9) and ROS production (Fig 10) Our data showed that 376
exogenous t180-P or d180 alone did not affect cell death or ROS production a 377
finding consistent with data obtained by Coursol et al (2015) In t180-P-treated 378
WT plants symptoms and ion leakage triggered by B cinerea or Pst infection 379
was significantly reduced (Fig 9 A C and E) Exogenously applied d180 did 380
not modify disease symptoms and electrolyte leakage in WT-infected plants by 381
B cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst 382
strain (Fig 9 B D and F) Interestingly disease symptoms and electrolyte 383
leakage were strongly reduced when WT plants were co-infiltrated with d180 384
and Pst AvrRPM1 (Fig 9 B and F) 385
Whereas addition of t180-P increased and delayed ROS production upon 386
challenge with B cinerea it reduced the Pst-induced oxidative burst (Fig 10 A-387
C) d180 had no significant effect on ROS accumulation triggered by B cinerea 388
(Fig 10D) However it dramatically reduced the Pst-induced oxidative burst 389
(Fig 10 E and F) These data indicate that exogenously applied t180-P and 390
d180 modify signaling event and cell death triggered by infection with these two 391
pathogens 392
393
SA and ETJA signaling pathways are modified in Atdpl1-1 mutant plants 394
after pathogen challenge 395
Disruption of sphingolipid contents between WT and Atdpl1 plants could result 396
in differential activation of defense responses after pathogen infection PR1 and 397
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
PR5 are well-known SA-dependent defense marker gene Nonexpressed 398
Pathogen Related1 (NPR1) was shown to be a key regulator of SA-mediated 399
suppression of JA signaling (Spoel et al 2003) PDF12 CHIT and ERF1 400
expression is regulated by JA and ET whereas VSP1 and JAZ8 are mostly 401
responsive to JA (Glazebrook 2005 Pieterse et al 2009) First the expression 402
pattern of these defense genes was monitored in WT and Atdpl1-1 mutant 403
plants No significant difference in expression of these defense genes was 404
detected in WT and Atdpl1-1 mutant plants grown under standard conditions 405
(Fig 11 and 12) These results indicated that inactivation of the gene encoding 406
LCB-P lyase itself did not result in any defense response changes in plants 407
The expression levels of defense-related genes in Atdpl1-1 mutant plants were 408
then compared to WT plants in response to B cinerea infection (Fig 11) 409
Whereas PR1 PR5 NPR1 and VSP1 expressions showed similar induction 410
levels in both genotypes expression of PDF12 CHIT ERF1 and JAZ8 was 411
markedly enhanced in Atdpl1-1 mutant compared to WT plants At 48 hpi there 412
was a 12-fold increase for PDF12 and a 2-fold increase for CHIT ERF1 and 413
JAZ8 compared to the WT plants (Fig 11) Since JA responsive genes were up-414
regulated in Atdpl1-1 mutant expression of three genes encoding key enzymes 415
in JA biosynthesis LOX2 AOC2 and OPR3 and JAR1 encoding the enzyme 416
that converts JA to the jasmonoyl- isoleucine (JA-Ile) conjugate (Staswick and 417
Tiryaki 2004) was also followed Results showed that LOX2 and AOC2 were 418
significantly up-regulated up to 24 hpi in Atdpl1-1 mutant but transcripts 419
returned to a level comparable to the WT thereafter (Fig 11) In contrast 420
expression of OPR3 was similar in both genotypes JAR1 expression was not 421
affected by the fungus inoculation (Fig11) These results indicated that both JA 422
synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants 423
When infected with Pst WT plants displayed a strong induction of PR1 424
expression and as expected this induction was more pronounced (4-fold at 48 425
hpi) in the case of incompatible interaction (Fig 12) Surprisingly a significant 426
repression of this gene was observed 30 hpi in Atdpl1-1 mutant compared to 427
WT plants (x6 for Pst DC3000 and x4 for Pst AvrRPM1) but level of PR1 428
expression was still higher in incompatible compared to compatible interaction 429
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
Accumulation of PR5 transcripts was also slightly more important in WT plants 430
but expression levels were more important in the case of the compatible 431
interaction (Fig 12) Under Pst attack NPR1 was slightly induced but no 432
difference between WT and Atdpl1-1 mutant was observed CHIT expression 433
was also more induced in response to Pst AvrRPM1 (x 90) than Pst DC3000 434
(x20) in WT plants and this induction profile was similar in Atdpl1-1 mutant 435
plants (Fig 12) As already described inoculation with the bacterial pathogen 436
(Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF12 437
expression either in WT or in Atdpl1-1 mutant plants In contrast ERF1 and 438
JAZ8 were induced during Pst infection and VSP1 expression was slightly 439
induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 440
infection (Fig 12) Expression of these three genes was markedly enhanced in 441
Atdpl1-1 mutant compared to WT plants At the end of the time course VSP1 442
ERF1 and JAZ8 mRNA levels were two- three- and six-fold higher in Atdpl1-1 443
than in WT plants after infection with either virulent or avirulent strains 444
respectively Similarly to B cinerea infection JAR1 expression was not affected 445
by inoculation with Pst Regarding genes involved in JA biosynthetic pathway 446
LOX2 was repressed AOC2 was not induced during Pst challenge and OPR3 447
was slightly induced but not difference between the two genotypes was 448
observed (Fig 12) These data suggested that only JA signaling pathway is 449
positively affected in mutant plants upon challenge with Pst 450
To get further information on Atdpl1-1 mutant defense responses some 451
defense-related phytohormones were also quantified (Fig 13 A and B) No 452
change in phytohormone basal levels was observed between WT and Atdpl1-1 453
mutant plants (Fig 13 A and B) This implied that Atdpl1-1 mutant plants in 454
contrast to other mutants with modified sphingolipid contents does not display 455
high constitutive SA amounts (Greenberg et al 2000 Wang et al 2008 456
Ternes et al 2011 Koumlnig et al 2012) Following pathogen attack all 457
phytohormone levels were enhanced SA accumulation was essentially 458
unchanged in the mutant compared to WT plants whatever the pathogen 459
considered Interestingly levels of JA and its biologically active conjugate JA-460
Ile were two to three times higher in Atdpl1-1 mutant compared to WT plants 461
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
after B cinerea or Pst infection respectively However no difference in JA 462
levels between virulent and avirulent interaction was noticed but JA-Ile 463
accumulation was slightly higher in the case of the avirulent interaction in 464
Atdpl1-1 plants Together our data suggest that JA-dependent signaling 465
pathway is preferentially activated in Atdpl1-1 mutant in response to pathogen 466
infection 467
468
469
DISCUSSION 470
471
Only few papers described a connection between sphingolipid content PCD 472
and defense reactions during biotic stress (Berkey et al 2012) Furthermore 473
most of them focused on responses against (hemi)biotrophic pathogen the role 474
of sphingolipid in plant defense against necrotrophs being largely unsolved 475
(Rivas-San Vicente et al 2013 Bi et al 2014) Moreover nearly all studies 476
revealed basal sphingolipid levels and data of sphingolipid contents during 477
pathogen infection were often not available (Peer et al 2010 Bi et al 2014) 478
The present work described a comparison of sphingolipid content during 479
hemibiotrophic and necrotrophic infection In the present study we investigated 480
the consequences of the disruption of the sphingolipid profiles on plant 481
immunity responses such as cell death ROS production and signaling of plant 482
defense response during pathogen infection 483
484
Interplays between sphingolipids and PCD 485
Like in animal systems new emerging evidence showed that bioactive 486
sphingolipids play a critical role as modulators of plant PCD (Berkey et al 487
2012 Saucedo-Garcia et al 2015) Here sphingolipid content analyses 488
showed that infection by B cinerea or Pst triggered accumulation of some 489
species known to act in favour of cell survival (LCB-Ps and hCers) or cell death 490
(LCBs and Cers) Interestingly Atdpl1-1 mutant displayed higher levels of d180 491
in response to infiltration (Fig2) Moreover this LCB reduced Pst-induced cell 492
death and symptoms especially in the case of the incompatible interaction (Fig 493
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
9) Since a HR often contributes to resistance to (hemi)biotrophic pathogens 494
our results suggested that a modification in d180 levels could impact plant cell 495
death and thus resistance to such pathogens Recently Coursol et al (2015) 496
showed that addition of d180 had no significant effect on viability of cryptogein-497
treated cells indicating that distinct mechanisms of regulation are involved in 498
cell death of cell culture or plant tissue or after treatment by an elicitor or a 499
pathogen Necrotrophs are pathogens that derive nutrients from dead or dying 500
cells PCD including HR can be beneficial to this kind of pathogens and could 501
thus facilitate their infection and spread of disease (Govrin and Levine 2000 502
Mayer et al 2001 Govrin et al 2006) Plants that are less potent to activate 503
HR or with reduced cell death present enhanced tolerance to B cinerea 504
infection and vice-versa (Govrin and Levine 2000 van Baarlen et al 2007) 505
Similarly antiapoptotic genes conferred resistance to necrotrophic fungi in 506
transgenic plants (Dickman et al 2001 El Oirdi and Bouarab 2007) A general 507
pattern established that infection of Arabidopsis by B cinerea is promoted by 508
and requires an active cell death program in the host (van Kan 2006) and 509
resistance against this fungus depends on the balance between cell death and 510
survival (van Baarlen et al 2007) Interestingly the Cer-accumulating acd5 511
mutant or Cer-infiltrated plants were more susceptible to several Botrytis 512
species (van Baarlen et al 2004 van Baarlen et al 2007) Moreover myriocin 513
a potent inhibitor of serine palmitoyltransferase (SPT) the first enzyme of 514
sphingolipid biosynthesis had death-antagonistic effect during the B elliptica-515
lily interaction (van Baarlen et al 2004) This suggests that sphingolipid 516
metabolism is involved in cell death triggered by Botrytis species Cell death 517
activation could thus be disturbed in Atdpl1 plants leading to a higher 518
susceptibility towards (hemi)biotrophs and higher tolerance towards 519
necrotrophs In the present work B cinerea infection triggered Cer and LCB 520
accumulation in WT plants It is thus possible that the necrotrophic fungus 521
promoted plant PCD-inducing factors (eg sphingolipids) in order to facilitate its 522
penetration and spread inside plant cells However exogenous d180 did not 523
modify ion leakage in presence of B cinerea suggesting that this LCB alone is 524
not involved in such mechanism Sphingolipid analysis revealed that B cinerea-525
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
infected Atdpl1-1 plants accumulated more VLCFA-hCers and t180-P and 526
t181-P but less Cers and LCBs compared to WT plants Interestingly our data 527
showed that exogenous t180-P reduced B cinerea- and Pst-induced cell death 528
(Fig 9) Thus t180-P appears to be essential to modulate plant cell death and 529
thus plant resistance in response to pathogen infection Moreover it was 530
recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs thereby VLCFA-531
hCers were key factors in Bax inhibitor-1 (AtBI-1)-mediated cell death 532
suppression (Nagano et al 2012) These results confirmed that sphingolipids 533
play important role in plant defense responses and plant is able to adjust its 534
response by regulating a dynamic balance between cell death (eg HR)- or cell 535
survival-related sphingolipids However in contrast to infected Atdpl1 mutant 536
the fah1fah2 double mutant presented reduced amount of hCers and elevated 537
levels of Cers and LCBs but showed no lesion phenotype (Koumlnig et al 2012) 538
Thus it seems that the connection between sphingolipids and PCD is regulated 539
by a fine-tuned process and could thus be more complex than expected Other 540
parameters such as defense signaling pathways could be involved in such 541
mechanism 542
543
Interconnections between sphingolipids and defense mechanisms 544
Sphingolipids (eg LCBs and Cers) participate in the induction andor control of 545
plant cell death Moreover plant cell death processes such as HR are also 546
associated with plant defense or disease It is thus conceivable that some 547
sphingolipids play key role in plant innate immunity Recent studies brought to 548
light interconnections between sphingolipids and defense mechanisms 549
Resistance to biotrophic pathogen often required ROS production (Torres et al 550
2002) Consistent with this Pst-infected Atdpl1-1 mutant displayed a reduced 551
accumulation of ROS and were more sensitive to the bacterial attack In 552
addition Atdpl1-1 mutant accumulated more d180 in response to infiltration 553
(Fig 2) and d180 strongly reduced ROS production upon challenge with this 554
bacterium (Fig 10) B cinerea-infected Atdpl1-1 plants displayed a higher 555
production of ROS (Fig 8) Several studies demonstrated that resistance 556
against B cinerea (and other necrotrophs) is accompanied by generation of 557
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
ROS and mutants impaired in ROS production failed to resist to the 558
necrotrophic pathogen (Contreras-Cornejo et al 2011 Kraepiel et al 2011 559
LHaridon et al 2011 Rasul et al 2012 Savatin et al 2014 Zhang et al 560
2014) It has been shown that LCBs but not LCB-Ps alone are able to induce 561
ROS production (Peer et al 2011) In the present study exogenously applied 562
t180-P increased B cinerea-induced ROS generation (Fig 10) Accordingly 563
cryptogein-induced ROS accumulation is enhanced by a pretreatment with 564
some LCB-Ps especially t180-P (Coursol et al 2015) This suggests that 565
sphingolipids may differently interact with ROS production depending on the 566
presence or not of an elicitor or pathogen Interestingly the similarity of ROS 567
accumulation upon infection between Atdpl1-1 plants and t180-P- or d180-568
treated WT plants indicated that phytosphingosine-1-phosphate and 569
dihydrosphingosine could have a key role in pathogen perception and thus in 570
plant resistance towards hemibiotrophic and necrotrophic pathogen 571
Several lines of evidence showed that plants disrupted in sphingolipid 572
metabolism often displayed spontaneous enhanced SA pathway (Greenberg et 573
al 2000 Brodersen et al 2002 Wang et al 2008 Ternes et al 2011 Koumlnig 574
et al 2012 Mortimer et al 2013 Rivas-San Vicente et al 2013 Wu et al 575
2015) Recently it has been shown that SA and its analog BTH 576
(benzothiadiazole) affect sphingolipid metabolism (Shi et al 2015) including 577
AtDPL1 gene expression (Wang et al 2006) Since activation of SA-dependent 578
pathway is effective against biotrophic and hemibiotrophic pathogens it has 579
been postulated that sphingolipids played a key role in defense against such 580
pathogens in an SA-dependent pathway (Sanchez-Rangel et al 2015) 581
However whereas acd5 erh1 and the double mutant fah1fah2 exhibited 582
enhanced resistance to powdery mildew they displayed a similar phenotype to 583
WT plants upon infection with P syringae pv maculicola or Verticillium 584
longisporum (Wang et al 2008 Koumlnig et al 2012) This suggests that SA 585
sphingolipid-triggered cell death and plant resistance could be independent 586
regarding the plantpathogen pair Unfortunately only basal levels of 587
sphingolipid were described no sphingolipid quantification during pathogen 588
infection is available making difficult a direct link between sphingolipid 589
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
metabolism and plant defense In the present work infection with either 590
necrotrophic or hemibiotrophic pathogen induced production of all quantified 591
phytohormones It has been reported that several pathogens including B 592
cinerea or Pst activated both SA and JA accumulation (Zimmerli et al 2001 593
Govrin and Levine 2002 Schmelz et al 2003 Spoel et al 2003 Block et al 594
2005 Glazebrook 2005 Veronese et al 2006) and cross-talk is thus used by 595
the plant to adjust its response in favor of the most effective pathway 596
Interestingly SPT-silenced tobacco plants displayed higher basal SA levels and 597
were more susceptible to A alternata infection However no information 598
concerning SA JA or sphingolipid levels in response to infection is available 599
especially as transgenic plants still displayed residual NbLCB2 gene expression 600
(Rivas-San Vicente et al 2013) In Arabidopsis acd5 mutant displayed 601
constitutive high SA levels and expression of PR1 gene This mutant was also 602
more susceptible to B cinerea and contained higher Cer levels but reduced 603
apoplastic ROS and PR1 and CHIT transcript accumulation upon infection 604
(Greenberg et al 2000 Bi et al 2014) Consistent with this Atdpl1-1 mutant 605
plants displayed similar Cer levels and PR1 expression higher apoplastic ROS 606
accumulation and CHIT up-regulation in response to infection but was more 607
resistant to the necrotrophic fungus In Arabidopsis it is now well admitted that 608
SA has antagonistic effect on JA signaling and reciprocally (Bostock 2005 609
Glazebrook 2005 Spoel et al 2007 Thaler et al 2012 Derksen et al 2013) 610
In tomato B cinerea produces an exopolysaccharide that activates the SA 611
pathway which through NPR1 antagonizes the JA signaling pathway thereby 612
allowing the fungus to enhance its disease (El Oirdi et al 2011) Moreover 613
NPR1 needs to be activated by SA (Cao et al 1998 Spoel et al 2003) Here 614
SA accumulated in WT plants and NPR1 was also stimulated upon infection 615
with B cinerea However SA signaling pathway was similar in Atdpl1-1 plants 616
Moreover JA biosynthetic and signaling pathways were enhanced in Atdpl1-1 617
mutant in response to B cinerea inoculation In Atdpl1-1 mutant it thus seems 618
that perturbation in sphingolipid metabolism rendered either SA unable to 619
activate NPR1 or NPR1 unable to antagonize JA accumulation Thus our 620
results highlighted that disturbance of sphingolipid metabolism could impact not 621
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
only cell death program but also JA signaling pathway leading to plant 622
tolerance towards necrotrophic pathogen such as B cinerea In that case the 623
relationship between sphingolipids and JA could be either indirect implying that 624
changes in sphingolipids operate in the crosstalk between SA and JA pathways 625
but in a NPR1 independent manner or direct as some key genes involved in JA 626
biosynthesis are up-regulated in Atdpl1-1 plants Similarly to B cinerea virulent 627
strain of Pst via its toxin coronatine exerts its virulence by stimulating JA 628
signaling pathway in order to inhibit SA signaling pathway and thus facilitate its 629
growth and development (Zhao et al 2003 Brooks et al 2005 Laurie-Berry et 630
al 2006 Uppalapati et al 2007 Geng et al 2012 Zheng et al 2012 Xin and 631
He 2013) The dramatic reduction in the expression of the SA-dependent 632
marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by over-633
accumulation of jasmonates in these plants Whereas VSP1 and JAZ8 634
expression profile correlated JA and JA-Ile accumulation profile in response to 635
infection with virulent or avirulent Pst PDF12 and CHIT expression did not 636
PDF12 and CHIT require both JA and ET signaling pathways but also the 637
function of MPK4 as JA-treated mpk4 mutants fail to express PDF12 (Petersen 638
et al 2000) Discrepancy between PDF12 expression and JA accumulation 639
has also been observed during the induced systemic resistance triggered by P 640
fluorescens and which is regulated through JA signaling pathway (van Wees et 641
al 1999) This suggested that a component in JA or ET signaling pathway 642
might be deficientnon functional in Atdpl1-1 mutant plants in response to Pst 643
infection or defense against Pst in Atdpl1-1 mutant might be regulated through 644
a pathway that does not include PDF12 or CHIT Collectively our results 645
suggested that AtDPL1 could be a negative andor a positive regulator of JA- 646
and SA-regulated defense pathway respectively Whereas relationship 647
between SA signaling and sphingolipids was often described (Sanchez-Rangel 648
et al 2015) our results highlight for the first time that sphingolipids could also 649
play a key role in JA signaling pathway 650
651
In conclusion we proposed a model in which plant cells of Atdpl1 mutant select 652
the most appropriate response to defend themselves against pathogen attack 653
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
by acting on sphingolipid metabolism in order to modulate the cell 654
deathsurvival balance in close cooperation with JA andor SA signaling 655
pathways (Fig 14) Whereas SA involvement in PCD is well known the 656
relationship between JA and cell death is less understood Plants treated with 657
coronatine which shares structural similarities with JA-Ile and functional 658
similarities with JA develop chlorosis (Bender et al 1999 Overmyer et al 659
2003) Coronatine-deficient mutants of Pst DC3000 are reduced in disease-660
associated necrosis and chlorosis (Brooks et al 2004 Brooks et al 2005) It 661
has been reported that JA is also essential in FB1- and AAL-induced cell death 662
(Asai et al 2000 Zhang et al 2011) Interestingly Atdpl1 mutant is more 663
sensitive to FB1 treatment (Tsegaye et al 2007) Thus sphingolipid 664
metabolism seemed to be intimately connected to defense processes to 665
regulate plant responses to biotic stresses In Arabidopsis MPK6 which is 666
involved in plant defense response (Ren et al 2008 Beckers et al 2009) has 667
recently been described as an important contributor to the LCB-mediated PCD 668
(Saucedo-Garcia et al 2011) However the deciphering of the precise pathway 669
leading to sphingolipid-induced cell death is far from being totally elucidated 670
Further identification of target genes and their functions will provide new 671
insights into how sphingolipids could be linked to cell death and defense 672
processes 673
674
675
676
MATERIALS AND METHODS 677
678
Chemicals 679
Phytosphingosine-1-phosphate (t180-P) and dihydrosphingosine (d180) were 680
purchased from Avanti Polar Lipids (Alabaster AL USA) Stock solutions were 681
prepared in ethanolDMSO (21 vv) (t180-P) or ethanol (d180) and dissolved 682
to a final concentration of 100 microM Luminol and horseradish peroxidase were 683
obtained from Sigma-Aldrich (France) 684
685
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
Plant material and growth conditions 686
Seeds of the Arabidopsis SALK lines 020151 (referred to as Atdpl1-1) 093662 687
(Atdpl1-2) and 078119 (Atdpl1-3) containing a T-DNA insertion in the 688
At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Center 689
(NASC httparabidopsisinfo) SALK_020151 mutant was chosen for the 690
performed experiments since it exhibits same phenotype than other Atdpl1 691
mutants but displays a complete lack of mRNA and a higher LCBLCB-P 692
accumulation in response to FB1 treatment (Tsegaye et al 2007) Mutant and 693
wild-type (Col-0) plants were grown and maintained under 12 h light12h dark 694
conditions (150 μmol m-2 sec-1 20degC and 60 humidity) for 35 days 695
696
Isolation of T-DNA insertion mutant and genotype characterization 697
The mutant SALK_020151 SALK_093662 (Tsegaye et al 2007) and 698
SALK_078119 were isolated according to the published procedure SIGnAL 699
(Alonso et al 2003) Genotype of the knockout mutant line was analyzed by 700
PCR reactions using primers specific for the AtDPL1 gene (forward 5rsquo-701
AGAAAGGCCTCAAAGCTTGTC-3rsquo and reverse 5rsquo-702
TGCCAAATAGCATCATTCCTC-3rsquo) and primer specific for the T-DNA (LB1a 5-703
TGGTTCACGTAGTGGGCCATCG-3) 704
705
Sphingolipidomic analysis 706
Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by 707
LC-ESI-MSMS were performed as described (Markham and Jaworski 2007) 708
with modifications using a Shimadzu Prominence UHPLC system and a 709
4000QTRAP mass spectrometer (AB SCIEX) Sphingolipids were separated on 710
a 100mm Dionex Acclaim C18 column Data analysis was performed using 711
Analyst 16 and Multiquant 21 software (AB SCIEX) Four to five biologically 712
independent repeats were performed and a minimum of three technical 713
replicates was run from each sample 714
715
RNA extraction and real-time quantitative RT-PCR 716
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
Isolation of total RNA and real-time PCR was performed as described in Le 717
Henanff et al (2013) Gene specific primers are described in Supplemental 718
Table S1 For each experiment PCR reactions were performed in duplicate and 719
at least 3 independent experiments were analyzed Transcript levels were 720
normalized against those of the Actin gene as an internal control Fold induction 721
compared to mock treated sample was calculated using the ΔΔ Ct method 722
723
Pathogen growth and inoculation 724
B cinerea strain B0510 was grown on solid tomato medium (tomato juice 25 725
(vv) agar 25 (pv)) during 21 days at 22degC Collected conidia were 726
resuspended in potato dextrose broth (PDB) supplemented by Sylwett L-77 727
002 to a final density of 105 conidia mL-1 After incubation 3 h at 22degC and 728
150 rpm germinated spores were used for plant inoculation by spraying the 729
upper face of the leaves Control inoculations were performed with PDB Sylwett 730
L-77 002 731
The bacterial leaf pathogen Pst strain DC3000 (Pst DC3000) or Pst carrying 732
AvrRPM1 (Pst AvrRPM1) were cultured overnight at 28degC in liquid Kingrsquos B 733
medium supplemented with rifampicin (50 microg mL-1) and kanamycin (50 microg mL-734 1) Subsequently bacterial cells were collected by centrifugation and 735
resuspended in 10 mM MgCl2 to a final density of 107 colony forming units (cfu) 736
mL-1 (optical density = 001) The bacterial solutions were thus infiltrated from 737
the abaxial side into leaf using a 1-mL syringe without a needle Control 738
inoculations were performed with 10 mM MgCl2 739
Leaves were collected from 0 to 48 hpi and frozen in liquid nitrogen and stored 740
at - 80degC until use 741
742
Pathogen assay in planta 743
B cinerea infections were performed as previously described (Le Heacutenanff et al 744
2013) Plants were placed in translucent boxes under high humidity at 150 microE 745
m-2 s-1 Five or six leaves per plants were droplet-inoculated with 5 microL of the 746
conidia suspension adjusted at 105 conidia mL-1 in PDB Lesion diameters were 747
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
measured 48 and 60 hpi Forty to 60 leaves were inoculated per treatment and 748
per genotype and experiments were independently repeated 4 times 749
Bacterial infections were performed as previously described (Sanchez et al 750
2012) Briefly 8 foliar discs from 4 leaves were excised using a cork borer and 751
ground in 1 mL MgCl2 (10 mM) with a plastic pestle Appropriate dilutions were 752
plated on Kingrsquos B medium with appropriate antibiotics and bacterial colonies 753
were counted Data are reported as means and SD of the log (cfu cm-2) of three 754
replicates Growth assays were performed four times with similar results 755
756
Electrolyte leakage 757
Ten minutes after bacteria injection (Torres et al 2002) or 20 h after B cinerea 758
(Govrin and Levine 2002) infection 9-mm-diameter leaf discs were collected 759
from the infected area and washed extensively with water for 50 min and then 760
eight discs were placed in a tube with 15 mL of fresh water To test sphingolipid 761
effect on ion leakage pathogen inoculum was supplemented or not with 100 microM 762
t180-P or d180 Conductivity measurements (3ndash4 replicates for each 763
treatment) were then measured over time by using a B-771 LaquaTwin (Horiba) 764
conductivity meter 765
766
ROS production 767
Measurements of ROS production were performed as described previously 768
(Smith and Heese 2014) Briefly single leaf disc halves were placed in wells of 769
a 96-well plate containing 150 μL of distilled water and then incubated overnight 770
at room temperature Just before ROS quantification distilled water was 771
replaced by 150 μL of an elicitation solution containing 20 μg mL-1 horseradish 772
peroxidase and 02 μM luminol For tests involving bacteria Pst was added to 773
the elicitation solution to a final bacterial concentration of 108 cfu mL-1 For tests 774
involving B cinerea germinated spores were added to the elicitation solution to 775
a final density of 105 conidia mL-1 For tests involving sphingolipids 100 microM 776
t180-P or d180 were added concomitantly with bacterium or fungus to the 777
elicitation solution 778
779
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
Phytohormone analysis 780
Phytohormones were quantified using UHPLC-MSMS according to Glauser et 781
al (2014) 782
783
Supplemental material 784
Supplemental Figure S1 Time-course of AtDPL1 and RbcS expression after B 785
cinerea or Pst infection 786
Supplemental Figure S2 Glucosylceramide contents after B cinerea or Pst 787
infection 788
Supplemental Figure S3 Total content in major sphingolipid classes in WT and 789
Atdpl1-1 mutant plants before infection with B cinerea or Pst 790
Supplemental Figure S4 Total content in major sphingolipid classes in WT and 791
Atdpl1-1 mutant plants after infection with B cinerea or Pst 792
Supplemental Table S1 Gene-specific primers used in real time reverse-793
transcription polymerase chain reaction 794
795
ACKNOWLEDGEMENTS 796
We thank Gaetan Glauser and Neil Villard from the Neuchacirctel Platform of 797
Analytical Chemistry (NPAC) (University of Neuchacirctel Switzerland) for excellent 798
technical assistance in phytohormone quantification 799
800
FIGURE LEGENDS 801
802
Figure 1 Atdpl1 mutants are more tolerant to B cinerea but more 803
susceptible to Pst than WT 804
B cinerea conidia suspension was deposited by using droplet-inoculation (A 805
and B) or spray-inoculation (E) on leaves of WT and Atdpl1 mutant plants Pst 806
solution was infiltrated in WT and Atdpl1 mutant leaves (A C and D) A 807
Photographs represent disease symptoms observed 60 or 72 h after infection 808
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
by the fungus or Pst respectively B Symptoms due to B cinerea infection 809
were scored by defining three lesion diameter (d) classes dlt7 mm 7ledle9 mm 810
dgt9 mm (48 hpi) dlt10 mm 10ledle12 mm dgt12 mm (60 hpi) Statistical 811
differences of the mean of lesion diameter between WT and Atdpl1 plants were 812
calculated with a Kruskal-Wallis test with Plt0005 C Bacterial growth of 813
virulent Pst strain DC3000 and D avirulent Pst strain AvrRPM1 at 0 6 24 48 814
and 54 hpi E B cinerea and Pst growth was quantified by qRT-PCR 3 and 48 815
h after pathogen infection in leaves of WT and Atdpl1 mutant plants Asterisks 816
indicate significant differences between WT-and Atdpl1-treated samples 817
according to Studentrsquos t test (Plt0005) Results are representative of three 818
independent experiments 819
820
Figure 2 Free LCB and LCB-P accumulation after challenge with 821
pathogen 822
Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore 823
suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst 824
AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and 825
LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant 826
differences between the pathogen-treated WT sample and the control sample 827
and asterisks on Atdpl1-1 bars indicate significant differences between the 828
pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 829
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the 830
mean of four to five independent biological experiments plusmn SD Notice the 831
different scale of LCB-P levels between WT and Atdpl1-1 plants 832
833
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or 834
Pst infection 835
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 836
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 837
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst 838
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT 839
bars indicate significant differences between the pathogen-treated WT sample 840
and the control sample and asterisks on Atdpl1-1 bars indicate significant 841
differences in total species between the pathogen-treated WT sample and the 842
pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 843
Plt001 Plt0005) Asterisks have only been considered for the total species 844
displaying the same fatty acid or hydroxylationunsaturation degree Results are 845
the mean of four to five independent biological experiments plusmn SD 846
847
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants 848
upon pathogen infection 849
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 850
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 851
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 852
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 853
significant differences between the pathogen-treated WT sample and the 854
control sample and asterisks on Atdpl1-1 bars indicate significant differences 855
between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 856
sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) 857
Asterisks have only been considered for the total species displaying the same 858
fatty acid or hydroxylationunsaturation degree Results are the mean of four to 859
five independent biological experiments plusmn SD 860
861
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant 862
plants upon pathogen infection 863
Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed 864
with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or 865
infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 866
(I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate 867
significant differences between the pathogen-treated sample and the control 868
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
sample and asterisks on Atdpl1-1 bars indicate significant differences between 869
the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample 870
according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have 871
only been considered for the total species displaying the same fatty acid or 872
hydroxylationunsaturation degree Results are the mean of four to five 873
independent biological experiments plusmn SD 874
875
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen 876
inoculation 877
Conductivity (μS cm-1) of solution containing leaf discs from either WT or 878
Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst 879
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (B) Each value represents the mean 880
plusmn SD of three replicates per experiment The experiment was repeated three 881
times with similar results 882
883
Figure 7 Time-course of programmed cell death marker gene expression 884
after B cinerea or Pst infection 885
Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) 886
(A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst 887
AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one 888
representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C 889
and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 890
mutant plants with five biological replicates with comparable results 891
892
Figure 8 Transient ROS production in response to pathogen infection in 893
WT and Atdpl1-1 mutant plants 894
Time-course of ROS production in WT and Atdpl1-1 mutant plants in response 895
to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks 896
were immersed in a solution containing either 105 spores mL-1 of B cinerea or 897
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological 898
repetitions Three independent experiments were performed with similar results 899
900
Figure 9 Exogenously effect of phytosphingosine-1-phosphate and 901
dihydrosphingosine on electrolyte leakage in response to pathogen 902
infection in WT plants 903
A and B B cinerea conidia suspension was deposited on leaves of WT and 904
Atdpl1-1 mutant plants 15 min after infiltration of either t18-0-P or d180 905
solution Pst and either t18-0-P or d180 solution were co-infiltrated in WT and 906
Atdpl1-1 leaves Photographs represent symptoms observed 60 or 72 h after 907
infection by the fungus or Pst respectively Conductivity (μS cm-1) of solution 908
containing t180-P- or d180-infiltrated leaf discs from WT inoculated by 909
spraying B cinerea or PDB (control) solution (C and D) or by infiltration of Pst 910
DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (E and F) Each value represents the 911
mean plusmn SD of three replicates per experiment The experiment was repeated 912
three times with similar results 913
914
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and 915
dihydrosphingosine on ROS production in response to pathogen infection 916
in WT plants 917
Time-course of ROS production in t180-1-P- or d180-treated WT plants in 918
response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C 919
and F) infection Leaf disks were immersed in a solution containing 100 microM of 920
t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of 921
Pst Error bars represent SE of the mean from 12 biological repetitions Three 922
independent experiments were performed with similar results 923
924
Figure 11 Expression levels of JA and SA pathway-associated genes in 925
WT and Atdpl1-1 mutant plants during B cinerea infection 926
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Results are expressed as the fold increase in the transcript level compared with 927
the untreated control (time 0 h) referred to as the times1 expression level Values 928
shown are means plusmn SD of duplicate data from one representative experiment 929
among five independent repetitions 930
931
Figure 12 Expression levels of JA and SA pathway-associated genes in 932
WT and Atdpl1-1 mutant plants during Pst infection 933
Results are expressed as the fold increase in the transcript level compared with 934
the untreated control (time 0 h) referred to as the times1 expression level Values 935
shown are means plusmn SD of duplicate data from one representative experiment 936
among five independent repetitions 937
938
Figure 13 Analysis of phytohormone accumulation in stressed WT and 939
Atdpl1-1 mutant plants 940
JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h 941
following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks 942
indicate significant differences between WT and Atdpl1-1 samples according to 943
Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn 944
SD from one representative experiment among five independent repetitions 945
946
Figure 14 Schematic overview of interconnections between sphingolipid 947
metabolism cell death and defense signaling pathways in Atdpl1 mutant 948
plants upon pathogen attack 949
Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer 950
and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid 951
metabolism may also indirectly modulate cell death through its tightly 952
connection (double-headed dashed arrow) as positive andor negative regulator 953
to jasmonate andor SA signaling pathways respectively Reduced cell death 954
and high levels of jasmonates could thus explain that Atdpl1 mutant plants are 955
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition 956
single-headed arrows indicate activation double-headed arrows indicate 957
unknown regulatory mechanism Ald aldehyde Ethan-P 958
phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P 959
Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine 960
kinase 1 961
962
963
LITERATURE CITED 964
Abbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E 965 Merrill AH Jr Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of 966 sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 967 1085-1093 968
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ 969 (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like 970 programmed cell death in plants Biochem Biophys Res Commun 410 574-580 971
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK 972 Zimmerman J Barajas P Cheuk R Gadrinab C Heller C Jeske A Koesema E 973 Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N 974 Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-975 Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw E Brogden D 976 Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional 977 mutagenesis of Arabidopsis thaliana Science 301 653-657 978
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin 979 B1-induced cell death in arabidopsis protoplasts requires jasmonate- ethylene- and 980 salicylate-dependent signaling pathways Plant Cell 12 1823-1836 981
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 982 473-488 983
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) 984 Mitogen-activated protein kinases 3 and 6 are required for full priming of stress 985 responses in Arabidopsis thaliana Plant Cell 21 944-953 986
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode 987 of action regulation and biosynthesis by peptide and polyketide synthetases Microbiol 988 Mol Biol Rev 63 266-292 989
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary 990 metabolism and plant-pathogen interactions J Exp Bot 58 4019-4026 991
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death 992 connection and beyond Front Plant Sci 3 68 993
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg 994 JT Su WW Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses 995 and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 996 3449-3467 997
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to 998 virulent bacterial pathogens in tomato Plant Physiol 138 1481-1490 999
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe 1000 Interact 22 487-497 1001
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost 1002 and benefit Annu Rev Phytopathol 43 545-580 1003
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ 1004 (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria 1005 alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-1006 4966 1007
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE 1008 Mundy J (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a 1009 sphingosine transfer protein causes activation of programmed cell death and defense 1010 Genes Dev 16 490-502 1011
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine 1012 promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis 1013 thaliana Mol Plant Pathol 6 629-639 1014
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A 1015 Rangaswamy V Penaloza-Vazquez A Bender CL Kunkel BN (2004) Identification 1016 and characterization of a well-defined series of coronatine biosynthetic mutants of 1017 Pseudomonas syringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174 1018
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by 1019 overexpression of an essential regulatory gene in systemic acquired resistance Proc 1020 Natl Acad Sci U S A 95 6531-6536 1021
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain 1022 base hydroxylation is important for growth and regulation of sphingolipid content and 1023 composition in Arabidopsis Plant Cell 20 1862-1878 1024
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell 1025 Death Differ 18 1247-1256 1026
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-1027 Bucio J (2011) Trichoderma-induced plant immunity likely involves both hormonal- and 1028 camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance 1029 against necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563 1030
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J 1031 Simon-Plas F (2015) Long-chain bases and their phosphorylated derivatives 1032 differentially regulate cryptogein-induced production of reactive oxygen species in 1033 tobacco (Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249 1034
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia 1035 amylovora pathogenicity Mol Plant Microbe Interact 11 734-742 1036
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance 1037 Plant Sci 207 79-87 1038
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of 1039 disease development in plants expressing animal antiapoptotic genes Proc Natl Acad 1040 Sci U S A 98 6957-6962 1041
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M 1042 Huang X Lyons BM Hein PP Gillaspy GE (2010) The Arabidopsis thaliana Myo-1043 inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and 1044 suppression of cell death Plant Cell 22 888-903 1045
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to 1046 understanding sphingolipid metabolism in Arabidopsis thaliana Ann Bot 93 483-497 1047
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease 1048 caused by the necrotrophic pathogen Botrytis cinerea New Phytol 175 131-139 1049
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A 1050 Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between 1051 immune pathways to promote disease development in tomato Plant Cell 23 2405-2421 1052
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas 1053 syringae is a multifunctional suppressor of Arabidopsis defense Plant Cell 24 4763-1054 4774 1055
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic 1056 pathogens Annu Rev Phytopathol 43 205-227 1057
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas 1058 eds Arabidopsis protocols methods in molecular biology Vol 1062 Springer 1059 Netherlands pp 597ndash608 1060
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the 1061 necrotrophic pathogen Botrytis cinerea Curr Biol 10 751-757 1062
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis 1063 cinerea elicits various defense responses but does not induce systemic acquired 1064 resistance (SAR) Plant Mol Biol 48 267-276 1065
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from 1066 Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other 1067 plants and promotes the gray mold disease Phytopathology 96 299-307 1068
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death 1069 and defense-related responses from disease resistance in the Arabidopsis mutant 1070 acd5 Genetics 156 341-350 1071
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A 1072 duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and 1073 RPS2-mediated hypersensitive response Plant J 44 258-270 1074
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky 1075 P Lipka V Feussner I (2012) Arabidopsis mutants of sphingolipid fatty acid alpha-1076 hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097 1077
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844 1078 Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) 1079
Analysis of the plant bos1 mutant highlights necrosis as an efficient defence 1080 mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991 1081
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek 1082 HJ Hess S Mir R Leon J Lamotte O Metraux JP (2011) A permeable cuticle is 1083 associated with the release of reactive oxygen species and induction of innate 1084 immunity PLoS Pathog 7 e1002148 1085
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S 1086 Mazars C Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in 1087 tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318 1088
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A 1089 Bellec Y Faure JD Ranjeva R Mazars C (2010) Nuclear calcium controls the 1090 apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 1091 47 92-100 1092
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana 1093 JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-1094 dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe 1095 Interact 19 789-800 1096
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F 1097 Cordelier S Dhondt-Cordelier S (2013) Grapevine NAC1 transcription factor as a 1098 convergent node in developmental processes abiotic stresses and 1099 necrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893 1100
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate 1101 programmed cell death in plants Genes Dev 17 2636-2641 1102
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf 1103 senescence in Arabidopsis thaliana Physiol Plant 92 322-328 1104
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis 1105 thaliana by reversed-phase high-performance liquid chromatography coupled to 1106 electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom 21 1107 1304-1314 1108
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids 1109 function follows form Curr Opin Plant Biol 16 350-357 1110
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant 1111 pathogens in hosts expressing the hypersensitive response Phytochemistry 58 33-41 1112
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy 1113 AM Matsunaga T Kurz S Stephens E Baldwin TC Ishii T Napier JA Weber AP 1114 Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggers 1115 salicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894 1116
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the 1117 centenary is upon us but how much do we know J Exp Bot 59 501-520 1118
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) 1119 Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are 1120 functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant 1121 Physiol 159 1138-1148 1122
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal 1123 control of cell death Trends Plant Sci 8 335-342 1124
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent 1125 reactive oxygen species production in Arabidopsis leaves FEBS Lett 585 3006-3010 1126
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers 1127 de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana FEBS 1128 Lett 584 4053-4056 1129
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB 1130 Lacy M Austin MJ Parker JE Sharma SB Klessig DF Martienssen R Mattsson 1131 O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates 1132 systemic acquired resistance Cell 103 1111-1120 1133
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-1134 molecule hormones in plant immunity Nat Chem Biol 5 308-316 1135
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D 1136 Jeandroz S (2012) Nitric oxide production mediates oligogalacturonide-triggered 1137 immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 1138 35 1483-1499 1139
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive 1140 MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis Proc Natl Acad Sci U 1141 S A 105 5638-5643 1142
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid 1143 biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses 1144 and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-1145 136 1146
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and 1147 defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 1148 317-343 1149
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F 1150 Dorey S (2012) Rhamnolipids elicit defense responses and induce disease resistance 1151 against biotrophic hemibiotrophic and necrotrophic pathogens that require different 1152 signaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant 1153 Physiol 160 1630-1641 1154
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M 1155 Plasencia J (2015) Deciphering the link between salicylic acid signaling and 1156 sphingolipid metabolism Front Plant Sci 6 125 1157
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases 1158 phosphatidic acid MAPKs and reactive oxygen species as nodal signal transducers in 1159 stress responses in Arabidopsis Front Plant Sci 6 55 1160
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana 1161 S Markham JE Lozano-Rosas MG Dietrich CR Ramos-Vega M Cahoon EB 1162 Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serine 1163 palmitoyltransferase are required in the signaling pathway that mediates cell death 1164 induced by long chain bases in Arabidopsis New Phytol 191 943-957 1165
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The 1166 Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein 1167 kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1168 165 1188-1202 1169
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson 1170 JH 3rd (2003) Simultaneous analysis of phytohormones phytotoxins and volatile 1171 organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557 1172
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial 1173 signatures Annu Rev Plant Biol 63 451-482 1174
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect 1175 of salicylic acid on sphingolipid metabolism Front Plant Sci 6 186 1176
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K 1177 Hannun YA Zuo J (2007) Involvement of sphingoid bases in mediating reactive 1178 oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 1179 17 1030-1040 1180
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species 1181 production in Arabidopsis leave tissue in response to living Pseudomonas syringae 1182 Plant Methods 10 6 1183
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses 1184 against pathogens with different lifestyles Proc Natl Acad Sci U S A 104 18842-18847 1185
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala 1186 AJ Metraux JP Brown R Kazan K Van Loon LC Dong X Pieterse CM (2003) 1187 NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense 1188 pathways through a novel function in the cytosol Plant Cell 15 760-770 1189
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that 1190 conjugates it to isoleucine in Arabidopsis Plant Cell 16 2117-2127 1191
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R 1192 (2009) Unraveling the roles of sphingolipids in plant innate immunity Plant Signal 1193 Behav 4 536-538 1194
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I 1195 (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in 1196 Arabidopsis thaliana New Phytol 192 841-854 1197
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal 1198 crosstalk Trends Plant Sci 17 260-270 1199
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease 1200 signaling in Arabidopsis Curr Opin Immunol 13 63-68 1201
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429 1202 Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and 1203
AtrbohF are required for accumulation of reactive oxygen intermediates in the plant 1204 defense response Proc Natl Acad Sci U S A 99 517-522 1205
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG 1206 Chen M Cahoon EB Dunn TM (2007) Arabidopsis mutants lacking long chain base 1207 phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chain 1208 base phosphate J Biol Chem 282 28195-28206 1209
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and 1210 effector-triggered immunity Curr Opin Plant Biol 13 459-465 1211
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) 1212 The phytotoxin coronatine contributes to pathogen fitness and is required for 1213 suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas 1214 syringae pv tomato DC3000 Mol Plant Microbe Interact 20 955-965 1215
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the 1216 fungal pathogen Botrytis elliptica Mol Plant Pathol 5 559-574 1217
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis 1218 of host and non-host interactions of Arabidopsis with three Botrytis species an 1219 important role for cell death control Mol Plant Pathol 8 41-54 1220
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant 1221 Sci 11 247-253 1222
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) 1223 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not 1224 associated with a direct effect on expression of known defense-related genes but 1225 stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge Plant 1226 Mol Biol 41 537-549 1227
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H 1228 Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays 1229 distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens Plant 1230 Cell 18 257-273 1231
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory 1232 nodes in the transcriptional network of systemic acquired resistance in plants PLoS 1233 Pathog 2 e123 1234
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang 1235 GL Bellizzi M Parsons JF Morrissey D Bravo JE Lynch DV Xiao S (2008) An 1236 inositolphosphorylceramide synthase is involved in regulation of plant programmed cell 1237 death associated with defense in Arabidopsis Plant Cell 20 3163-3179 1238
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis 1239 ceramidase AtACER functions in disease resistance and salt tolerance Plant J 81 767-1240 780 1241
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for 1242 probing disease susceptibility and hormone signaling in plants Annu Rev Phytopathol 1243 51 473-498 1244
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of 1245 two rice long-chain base kinase genes and their function in disease resistance and cell 1246 death Mol Biol Rep 40 117-127 1247
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and 1248 ethylene in Alternaria alternata f sp lycopersici toxin-induced tomato cell death J Exp 1249 Bot 62 5405-5418 1250
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from 1251 Botrytis cinerea improves disease resistance in Arabidopsis thaliana Biotechnol Lett 1252 36 1069-1078 1253
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of 1254 Pseudomonas syringae pv tomato promote bacterial speck disease in tomato by 1255 targeting the jasmonate signaling pathway Plant J 36 485-499 1256
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) 1257 Coronatine promotes Pseudomonas syringae virulence in plants by activating a 1258 signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-1259 596 1260
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of 1261 Arabidopsis against the necrotrophic fungus Botrytis cinerea Plant Physiol 126 517-1262 523 1263
1264
1265
1266
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgD
ownloaded on M
ay 17 2021 - Published by C
opyright (c) 2020 Am
erican Society of Plant Biologists A
ll rights reserved
000
003
006
009
012
nmol
g-1
DW
Control Bc
0
2
4
6
8
nmol
g-1
DW
Control Bc
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 1 2 3 4 5 6 Control
DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
0 05
1 15
2 25
3 Control DC3000 AvrRPM1
A B C D
E F G H
WT WT Atdpl1-1 Atdpl1-1
Figure 2 Free LCB and LCB-P accumulation after challenge with pathogen Leaves of WT or Atdpl1-1 mutant plants were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A B E F) or infiltrated with Pst DC3000 Pst AvrRPM1 or MgCl2 (Control) (C D G H) Quantifications of LCBs (A-D) and LCBPs (E-H) were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Results are the mean of four to five independent biological experiments plusmn SD Notice the different scale of LCB-P levels between WT and Atdpl1-1 plants
0
2
4
6
8 Control Bc
0
05
1
15
2
25 Control Bc
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
A
C
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
d180 d181 t180 t181 0
50
100
d180 d181 t180 t181 0
50
100
0
50
100
nmol
g-1
DW
0
50
100
nmol
g-1
DW
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
d180 d181 t180 t181 0
50
100
150
0
50
100
150
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
50
100
150
Fatty acid
Figure 3 Glycosylinositolphosphoceramide contents after B cinerea or Pst infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) or Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences in total species between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
B
D
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
nmol
g-1
DW
d180 d181 t180 t181 0
1
2
3
4
5
d180 d181 t180 t181 0
20
40
0
20
40
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
10
20
30
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
20
40
Fatty acid
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
A B
C D
E F
G H
I J
WT Atdpl1-1
Control
Bc
Control
DC3000
AvrRPM1
Figure 4 Ceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated WT sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD
httpsplantphysiolorgDownloaded on May 17 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
0
2
4
6
8
nmol
g-1
DW
d180 d181 t180 t181 0
2
4
6
8
d180 d181 t180 t181 0
5
10
15
20
0 5
10 15
20
nmol
g-1
DW
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
0
5
10
15
nmol
g-1
DW
Fatty acid
d180 d181 t180 t181 0
10
20
Fatty acid
0
5
10
15
nmol
g-1
DW
d180 d181 t180 t181 0
5
10
15
A B
C D
E F
G H
I J
Control
Bc
Control
DC3000
AvrRPM1
WT Atdpl1-1
Figure 5 Hydroxyceramide species produced by WT and Atdpl1-1 mutant plants upon pathogen infection Leaves of WT (left panel) or Atdpl1-1 mutant (right panel) plants were sprayed with PDB (Control) (A and B) or B cinerea spore suspension (Bc) (C and D) or infiltrated with MgCl2 (Control) (E and F) Pst DC3000 (G and H) Pst AvrRPM1 (I and J) Quantifications were performed 48 hpi Asterisks on WT bars indicate significant differences between the pathogen-treated sample and the control sample and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated WT sample and the pathogen-treated Atdpl1-1 sample according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Asterisks have only been considered for the total species displaying the same fatty acid or hydroxylationunsaturation degree Results are the mean of four to five independent biological experiments plusmn SD httpsplantphysiolorgDownloaded on May 17 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Figure 6 Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation Conductivity (microS cm-1) of solution containing leaf discs from either WT or Atdpl1-1 mutant inoculated with B cinerea or PDB (control) solution (A) Pst DC3000 or Pst AvrRPM1 or 10 mM MgCl2 (control) (B) Each value represents the mean plusmn SD of three replicates per experiment The experiment was repeated three times with similar results
0
50
100
150
200
250
0 12 24 36 48
Con
duct
ivity
(microS
cm-1
) Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
A
B
0
50
100
150
200
250
300
0 6 12 18 24
Con
duct
ivity
(microS
cm-1
)
Hpi
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48 0
2000
4000
6000
8000
10000
12000
0 12 24 36 48 Rel
ativ
e ge
ne e
xpre
ssio
n
0
200
400
600
800
1000
0 12 24 36 48 0
50
100
150
200
250
300
350
0 12 24 36 48
Rel
ativ
e ge
ne e
xpre
ssio
n
0
2000
4000
6000
0 12 24 36 48
Rel
ativ
e ge
ne e
pres
sion
Control WT Bc WT Control Atdpl1-1 Bc Atdpl1-1
0
2000
4000
6000
8000
0 12 24 36 48
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Hpi Hpi
A B
C D
E F
FMO
SAG12
SAG13
Figure 7 Time-course of programmed cell death marker gene expression after B cinerea or Pst infection Leaves were sprayed with B cinerea spore suspension (Bc) or PDB (Control) (A C and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control) (B D and F) The mean values plusmn SD from one representative experiment are shown QRT-PCR of FMO (A and B) SAG13 (C and D) and SAG12 (E and F) expression were performed in WT and Atdpl1-1 mutant plants with five biological replicates with comparable results
0
500
1000
1500
2000
0 100 200 300 400
RLU
s
Time (min)
Control WT Bc WT Control Atdpl1 -1 Bc Atdpl1-1
0
50
100
150
200
250
300
350
0 30 60 90
RLU
s
Time (min)
Control WT DC3000 WT Control Atdpl1-1 DC3000 Atdpl1-1
0
50
100
150
200
250
0 20 40 60 80 100
RLU
s
Time (min)
Control WT AvrRPM1 WT Control Atdpl1-1 AvrRPM1 Atdpl1-1
A
B
C
Figure 8 Transient ROS production in response to pathogen infection in WT and Atdpl1-1 mutant plants Time-course of ROS production in WT and Atdpl1-1 mutant plants in response to B cinerea (A) or Pst DC3000 (B) or Pst AvrRPM1 (C) infection Leaf disks were immersed in a solution containing either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
200
400
600
800
1000
0 100 200 300 400
RLU
s
Time (min)
Control t180-P Bc Bc + t180-P
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control t180-P DC3000 DC3000+t180-P
0
50
100
150
200
0 30 60 90 Time (min)
Control t180-P AvrRPM1 AvrRPM1+t180-P
D E F
0
200
400
600
0 100 200 300 400
RLU
s
Time (min)
Control d180 Bc Bc + d180
0
50
100
150
200
250
300
350
0 30 60 90 Time (min)
Control d180 DC3000 DC3000+d180
0
50
100
150
200
0 30 60 90 Time (min)
Control d180 AvrRPM1 AvrRPM1+d180
A B C
Figure 10 Exogenously effect of phytosphingosine-1-phosphate and dihydrosphingosine on ROS production in response to pathogen infection in WT plants Time-course of ROS production in t180-1-P- or d180-treated WT plants in response to B cinerea (A and D) or Pst DC3000 (B and E) or Pst AvrRPM1 (C and F) infection Leaf disks were immersed in a solution containing 100 microM of t180-1-P or d180 and either 105 spores mL-1 of B cinerea or 108 cfu mL-1 of Pst Error bars represent SE of the mean from 12 biological repetitions Three independent experiments were performed with similar results
0
50
100
150
0 12 24 36 48
Figure 11 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during B cinerea infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
PR1
Rel
ativ
e ge
ne e
xpre
ssio
n PDF12 CHIT
0
20
40
60
80
0 12 24 36 48
0
2000
4000
6000
8000
0 12 24 36 48
0
2
4
6
0 12 24 36 48
VSP1
0
50
100
0 12 24 36 48
ERF1
0
100
200
300
0 12 24 36 48
JAZ8
0 2 4 6 8
10
0 12 24 36 48
LOX2
0 2 4 6 8
10
0 12 24 36 48
AOC2
0
5
10
15
0 12 24 36 48
OPR3
0
05
1
15
2
0 12 24 36 48
JAR1
0
2
4
6
0 12 24 36 48
PR5
0
2
4
6
0 12 24 36 48
NPR1
0 2 4 6 8 10 0 12 24 36 48
Control WT Control Atdpl1-1 Bc WT Bc Atdpl1-1
Hpi
Rel
ativ
e ge
ne e
xpre
ssio
n
0
500
1000
1500
0 12 24 36 48
PR1
0
05
1
15
0 12 24 36 48 0
100
200
300
0 12 24 36 48
0
5
10
15
0 12 24 36 48
VSP1
ERF1 CHIT
PDF12
0
25
50
0 12 24 36 48
0
05
1
15
2
0 12 24 36 48
LOX2
0
05
1
15
2
0 12 24 36 48
AOC2
0
5
10
0 12 24 36 48
OPR3
0
08
16
24
0 12 24 36 48
JAR1
0
400
800
1200
0 12 24 36 48
0
20
40
60
80
0 12 24 36 48
PR5
JAZ8
0
2
4
6
8
0 12 24 36 48
NPR1
0 500 1000 1500
Control WT DC3000 WT AvrRPM1 WT Control Atdpl1-1 DC3000 Atdpl1-1 AvrRPM1 Atdpl1-1
Figure 12 Expression levels of JA and SA pathway-associated genes in WT and Atdpl1-1 mutant plants during Pst infection Results are expressed as the fold increase in the transcript level compared with the untreated control (time 0 h) referred to as the times1 expression level Values shown are means plusmn SD of duplicate data from one representative experiment among five independent repetitions
Hpi
0
50
100
150
200
250
300
350
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi WT 30 hpi Atdpl1-1 30 hpi
0
200
400
600
800
1000
SA JA JA-Ile
ng g
FW
-1
WT 0 hpi Atdpl1-1 0 hpi DC3000 WT 30 hpi DC3000 Atdpl1-1 30 hpi AvrRPM1 WT 30 hpi AvrRPM1 Atdpl1-1 30 hpi
A
B
Figure 13 Analysis of phytohormone accumulation in stressed WT and Atdpl1-1 mutant plants JA JA-Ile and SA accumulation in WT and Atdpl1-1 mutant plants 0 h or 30 h following B cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection Asterisks indicate significant differences between WT and Atdpl1-1 samples according to Studentrsquos t test ( Plt005 Plt001 Plt0005) Values shown are means plusmn SD from one representative experiment among five independent repetitions
Figure 14 Schematic overview of interconnections between sphingolipid metabolism cell death and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack Upon disruption of AtDPL1 gene infected plants accumulate some LCB-P hCer and GIPC species thus reducing cell death In Atdpl1 mutant sphingolipid metabolism may also indirectly modulate cell death through its tightly connection (double-headed dashed arrow) as positive andor negative regulator to jasmonate andor SA signaling pathways respectively Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B cinerea but more susceptible to Pst Bars indicate inhibition single-headed arrows indicate activation double-headed arrows indicate unknown regulatory mechanism Ald aldehyde Ethan-P phosphoethanolamine FAH fatty acid hydroxylase LCBK LCB kinase LCB-P Pase LCB-P phosphatase LOH Lag One Homolog SPHK1 sphingosine kinase 1
LCBs
LCB-Ps
Cell death
Necrotrophic pathogen B cinerea
Hemibiotrophic pathogen Pst
JA JA-Ile SA
plant cell Sphingolipid metabolism
Cers
Ethan-P + Ald
hCers
LOH123 FAH12
LCBK SPHK1
LCB-P Pase
AtDPL1
GIPCs
tolerance susceptibility
Parsed CitationsAbbas HK Tanaka T Duke SO Porter JK Wray EM Hodges L Sessions AE Wang E Merrill AH Jr Riley RT (1994) Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases Plant Physiol 106 1085-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alden KP Dhondt-Cordelier S McDonald KL Reape TJ Ng CK McCabe PF Leaver CJ (2011) Sphingolipid long chain basephosphates can regulate apoptotic-like programmed cell death in plants Biochem Biophys Res Commun 410 574-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk R Gadrinab CHeller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt M Hazari N Hom E Karnes MMulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel D Carter DE Marchand T Risseeuw EBrogden D Zeko A Crosby WL Berry CC Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science301 653-657
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Asai T Stone JM Heard JE Kovtun Y Yorgey P Sheen J Ausubel FM (2000) Fumonisin B1-induced cell death in arabidopsisprotoplasts requires jasmonate- ethylene- and salicylate-dependent signaling pathways Plant Cell 12 1823-1836
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant Mol Biol 69 473-488Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Beckers GJ Jaskiewicz M Liu Y Underwood WR He SY Zhang S Conrath U (2009) Mitogen-activated protein kinases 3 and 6 arerequired for full priming of stress responses in Arabidopsis thaliana Plant Cell 21 944-953
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bender CL Alarcon-Chaidez F Gross DC (1999) Pseudomonas syringae phytotoxins mode of action regulation and biosynthesisby peptide and polyketide synthetases Microbiol Mol Biol Rev 63 266-292
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berger S Sinha AK Roitsch T (2007) Plant physiology meets phytopathology plant primary metabolism and plant-pathogeninteractions J Exp Bot 58 4019-4026
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Berkey R Bendigeri D Xiao S (2012) Sphingolipids and plant defensedisease the death connection and beyond Front Plant Sci3 68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi FC Liu Z Wu JX Liang H Xi XL Fang C Sun TJ Yin J Dai GY Rong C Greenberg JT Su WW Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts Plant Cell 26 3449-3467
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Block A Schmelz E ODonnell PJ Jones JB Klee HJ (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomatoPlant Physiol 138 1481-1490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bolton MD (2009) Primary metabolism and plant defense--fuel for the fire Mol Plant Microbe Interact 22 487-497Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bostock RM (2005) Signal crosstalk and induced resistance straddling the line between cost and benefit Annu Rev Phytopathol43 545-580
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brandwagt BF Mesbah LA Takken FL Laurent PL Kneppers TJ Hille J Nijkamp HJ (2000) A longevity assurance gene homologof tomato mediates resistance to Alternaria alternata f sp lycopersici toxins and fumonisin B1 Proc Natl Acad Sci U S A 97 4961-4966
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brodersen P Petersen M Pike HM Olszak B Skov S Odum N Jorgensen LB Brown RE Mundy J (2002) Knockout of Arabidopsisaccelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defenseGenes Dev 16 490-502
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Bender CL Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcomingsalicylic acid-dependent defences in Arabidopsis thaliana Mol Plant Pathol 6 629-639
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brooks DM Hernandez-Guzman G Kloek AP Alarcon-Chaidez F Sreedharan A Rangaswamy V Penaloza-Vazquez A Bender CLKunkel BN (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonassyringae pv tomato DC3000 Mol Plant Microbe Interact 17 162-174
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Cao H Li X Dong X (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene insystemic acquired resistance Proc Natl Acad Sci U S A 95 6531-6536
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen M Markham JE Dietrich CR Jaworski JG Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis Plant Cell 20 1862-1878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coll NS Epple P Dangl JL (2011) Programmed cell death in the plant immune system Cell Death Differ 18 1247-1256Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Contreras-Cornejo HA Macias-Rodriguez L Beltran-Pena E Herrera-Estrella A Lopez-Bucio J (2011) Trichoderma-induced plantimmunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistanceagainst necrotrophic fungi Botrytis cinerea Plant Signal Behav 6 1554-1563
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Coursol S Fromentin J Noirot E Briegravere C Robert F Morel J Liang YK Lherminier J Simon-Plas F (2015) Long-chain bases andtheir phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco(Nicotiana tabacum) BY-2 cells New Phytol 205 1239-1249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dellagi A Brisset MN Paulin JP Expert D (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity Mol PlantMicrobe Interact 11 734-742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Derksen H Rampitsch C Daayf F (2013) Signaling cross-talk in plant disease resistance Plant Sci 207 79-87Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dickman MB Park YK Oltersdorf T Li W Clemente T French R (2001) Abrogation of disease development in plants expressinganimal antiapoptotic genes Proc Natl Acad Sci U S A 98 6957-6962
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Donahue JL Alford SR Torabinejad J Kerwin RE Nourbakhsh A Ray WK Hernick M Huang X Lyons BM Hein PP Gillaspy GE
(2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppressionof cell death Plant Cell 22 888-903
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dunn TM Lynch DV Michaelson LV Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism inArabidopsis thaliana Ann Bot 93 483-497
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M Bouarab K (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogenBotrytis cinerea New Phytol 175 131-139
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El Oirdi M El Rahman TA Rigano L El Hadrami A Rodriguez MC Daayf F Vojnov A Bouarab K (2011) Botrytis cinerea manipulatesthe antagonistic effects between immune pathways to promote disease development in tomato Plant Cell 23 2405-2421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geng X Cheng J Gangadharan A Mackey D (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressorof Arabidopsis defense Plant Cell 24 4763-4774
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens Annu Rev Phytopathol43 205-227
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Glauser G Vallat A Balmer D (2014) Hormone profiling In JJ Sanchez-Serrano J Salinas eds Arabidopsis protocols methods inmolecular biology Vol 1062 Springer Netherlands pp 597-608
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinereaCurr Biol 10 751-757
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen Botrytis cinerea elicits various defenseresponses but does not induce systemic acquired resistance (SAR) Plant Mol Biol 48 267-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Govrin EM Rachmilevitch S Tiwari BS Solomon M Levine A (2006) An elicitor from Botrytis cinerea induces the hypersensitiveresponse in Arabidopsis thaliana and other plants and promotes the gray mold disease Phytopathology 96 299-307
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Greenberg JT Silverman FP Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the Arabidopsis mutant acd5 Genetics 156 341-350
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kawasaki T Nam J Boyes DC Holt BF 3rd Hubert DA Wiig A Dangl JL (2005) A duplicated pair of Arabidopsis RING-finger E3ligases contribute to the RPM1- and RPS2-mediated hypersensitive response Plant J 44 258-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koumlnig S Feussner K Schwarz M Kaever A Iven T Landesfeind M Ternes P Karlovsky P Lipka V Feussner I (2012) Arabidopsismutants of sphingolipid fatty acid alpha-hydroxylases accumulate ceramides and salicylates New Phytol 196 1086-1097
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Koornneef A Pieterse CM (2008) Cross talk in defense signaling Plant Physiol 146 839-844Pubmed Author and Title
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kraepiel Y Pedron J Patrit O Simond-Cote E Hermand V Van Gijsegem F (2011) Analysis of the plant bos1 mutant highlightsnecrosis as an efficient defence mechanism during D dadantiiArabidospis thaliana interaction PLoS One 6 e18991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
LHaridon F Besson-Bard A Binda M Serrano M Abou-Mansour E Balet F Schoonbeek HJ Hess S Mir R Leon J Lamotte OMetraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunityPLoS Pathog 7 e1002148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Amelot N Beziat C Briere C Cotelle V Graziana A Grat S Mazars C Thuleau P (2011)Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H(2)O(2) production Mol Plant 4 310-318
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lachaud C Da Silva D Cotelle V Thuleau P Xiong TC Jauneau A Briere C Graziana A Bellec Y Faure JD Ranjeva R Mazars C(2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells Cell Calcium 4792-100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Laurie-Berry N Joardar V Street IH Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required forsuppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae Mol Plant Microbe Interact 19 789-800
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Le Henanff G Profizi C Courteaux B Rabenoelina F Gerard C Clement C Baillieul F Cordelier S Dhondt-Cordelier S (2013)Grapevine NAC1 transcription factor as a convergent node in developmental processes abiotic stresses andnecrotrophicbiotrophic pathogen tolerance J Exp Bot 64 4877-4893
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liang H Yao N Song JT Luo S Lu H Greenberg JT (2003) Ceramides modulate programmed cell death in plants Genes Dev 172636-2641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lohman KN Gan S John MC Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana PhysiolPlant 92 322-328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry Rapid Commun Mass Spectrom21 1304-1314
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Markham JE Lynch DV Napier JA Dunn TM Cahoon EB (2013) Plant sphingolipids function follows form Curr Opin Plant Biol 16350-357
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mayer AM Staples RC Gil-ad NL (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing thehypersensitive response Phytochemistry 58 33-41
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mortimer JC Yu X Albrecht S Sicilia F Huichalaf M Ampuero D Michaelson LV Murphy AM Matsunaga T Kurz S Stephens EBaldwin TC Ishii T Napier JA Weber AP Handford MG Dupree P (2013) Abnormal glycosphingolipid mannosylation triggerssalicylic acid-mediated responses in Arabidopsis Plant Cell 25 1881-1894
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Mur LA Kenton P Lloyd AJ Ougham H Prats E (2008) The hypersensitive response the centenary is upon us but how much dowe know J Exp Bot 59 501-520
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nagano M Takahara K Fujimoto M Tsutsumi N Uchimiya H Kawai-Yamada M (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses Plant Physiol159 1138-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Overmyer K Brosche M Kangasjarvi J (2003) Reactive oxygen species and hormonal control of cell death Trends Plant Sci 8335-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Bach M Mueller MJ Waller F (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production inArabidopsis leaves FEBS Lett 585 3006-3010
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Peer M Stegmann M Mueller MJ Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis ofphytosphingosine from sphinganine in Arabidopsis thaliana FEBS Lett 584 4053-4056
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Petersen M Brodersen P Naested H Andreasson E Lindhart U Johansen B Nielsen HB Lacy M Austin MJ Parker JE SharmaSB Klessig DF Martienssen R Mattsson O Jensen AB Mundy J (2000) Arabidopsis map kinase 4 negatively regulates systemicacquired resistance Cell 103 1111-1120
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pieterse CM Leon-Reyes A Van der Ent S Van Wees SC (2009) Networking by small-molecule hormones in plant immunity NatChem Biol 5 308-316
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rasul S Dubreuil-Maurizi C Lamotte O Koen E Poinssot B Alcaraz G Wendehenne D Jeandroz S (2012) Nitric oxide productionmediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana Plant Cell Environ 351483-1499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ren D Liu Y Yang KY Han L Mao G Glazebrook J Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexinbiosynthesis in Arabidopsis Proc Natl Acad Sci U S A 105 5638-5643
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rivas-San Vicente M Larios-Zarate G Plasencia J (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamianaactivates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f sp lycopersici Planta 237 121-136
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert-Seilaniantz A Grant M Jones JD (2011) Hormone crosstalk in plant disease and defense more than just jasmonate-salicylate antagonism Annu Rev Phytopathol 49 317-343
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez L Courteaux B Hubert J Kauffmann S Renault JH Clement C Baillieul F Dorey S (2012) Rhamnolipids elicit defenseresponses and induce disease resistance against biotrophic hemibiotrophic and necrotrophic pathogens that require differentsignaling pathways in Arabidopsis and highlight a central role for salicylic acid Plant Physiol 160 1630-1641
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sanchez-Rangel D Rivas-San Vicente M de la Torre-Hernandez ME Najera-Martinez M Plasencia J (2015) Deciphering the link
between salicylic acid signaling and sphingolipid metabolism Front Plant Sci 6 125Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Gavilanes-Ruiz M Arce-Cervantes O (2015) Long-chain bases phosphatidic acid MAPKs and reactive oxygenspecies as nodal signal transducers in stress responses in Arabidopsis Front Plant Sci 6 55
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saucedo-Garcia M Guevara-Garcia A Gonzalez-Solis A Cruz-Garcia F Vazquez-Santana S Markham JE Lozano-Rosas MGDietrich CR Ramos-Vega M Cahoon EB Gavilanes-Ruiz M (2011) MPK6 sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in ArabidopsisNew Phytol 191 943-957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Savatin DV Bisceglia NG Marti L Fabbri C Cervone F De Lorenzo G (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity Plant Physiol 1651188-1202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schmelz EA Engelberth J Alborn HT ODonnell P Sammons M Toshima H Tumlinson JH 3rd (2003) Simultaneous analysis ofphytohormones phytotoxins and volatile organic compounds in plants Proc Natl Acad Sci U S A 100 10552-10557
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schwessinger B Ronald PC (2012) Plant innate immunity perception of conserved microbial signatures Annu Rev Plant Biol 63451-482
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi C Yin J Liu Z Wu JX Zhao Q Ren J Yao N (2015) A systematic simulation of the effect of salicylic acid on sphingolipidmetabolism Front Plant Sci 6 186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 171030-1040
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smith JM Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue inresponse to living Pseudomonas syringae Plant Methods 10 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Johnson JS Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestylesProc Natl Acad Sci U S A 104 18842-18847
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spoel SH Koornneef A Claessens SM Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Metraux JP Brown R Kazan K VanLoon LC Dong X Pieterse CM (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol Plant Cell 15 760-770
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Staswick PE Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine inArabidopsis Plant Cell 16 2117-2127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Takahashi Y Berberich T Kanzaki H Matsumura H Saitoh H Kusano T Terauchi R (2009) Unraveling the roles of sphingolipids inplant innate immunity Plant Signal Behav 4 536-538
Pubmed Author and TitleCrossRef Author and Title
Google Scholar Author Only Title Only Author and Title
Ternes P Feussner K Werner S Lerche J Iven T Heilmann I Riezman H Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana New Phytol 192 841-854
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thaler JS Humphrey PT Whiteman NK (2012) Evolution of jasmonate and salicylate signal crosstalk Trends Plant Sci 17 260-270Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thomma BP Penninckx IA Broekaert WF Cammue BP (2001) The complexity of disease signaling in Arabidopsis Curr OpinImmunol 13 63-68
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA (2010) ROS in biotic interactions Physiol Plant 138 414-429Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Torres MA Dangl JL Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation ofreactive oxygen intermediates in the plant defense response Proc Natl Acad Sci U S A 99 517-522
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsegaye Y Richardson CG Bravo JE Mulcahy BJ Lynch DV Markham JE Jaworski JG Chen M Cahoon EB Dunn TM (2007)Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-181 long chainbase phosphate J Biol Chem 282 28195-28206
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tsuda K Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity CurrOpin Plant Biol 13 459-465
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uppalapati SR Ishiga Y Wangdi T Kunkel BN Anand A Mysore KS Bender CL (2007) The phytotoxin coronatine contributes topathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringaepv tomato DC3000 Mol Plant Microbe Interact 20 955-965
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Staats M van Kan J (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica MolPlant Pathol 5 559-574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Baarlen P Woltering EJ Staats M van Kan J (2007) Histochemical and genetic analysis of host and non-host interactions ofArabidopsis with three Botrytis species an important role for cell death control Mol Plant Pathol 8 41-54
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Kan JA (2006) Licensed to kill the lifestyle of a necrotrophic plant pathogen Trends Plant Sci 11 247-253Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van Wees SC Luijendijk M Smoorenburg I van Loon LC Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance(ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates theexpression of the jasmonate-inducible gene Atvsp upon challenge Plant Mol Biol 41 537-549
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Veronese P Nakagami H Bluhm B Abuqamar S Chen X Salmeron J Dietrich RA Hirt H Mengiste T (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogensPlant Cell 18 257-273
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang D Amornsiripanitch N Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network ofsystemic acquired resistance in plants PLoS Pathog 2 e123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang W Yang X Tangchaiburana S Ndeh R Markham JE Tsegaye Y Dunn TM Wang GL Bellizzi M Parsons JF Morrissey DBravo JE Lynch DV Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed celldeath associated with defense in Arabidopsis Plant Cell 20 3163-3179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wu JX Li J Liu Z Yin J Chang ZY Rong C Wu JL Bi FC Yao N (2015) The Arabidopsis ceramidase AtACER functions in diseaseresistance and salt tolerance Plant J 81 767-780
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xin XF He SY (2013) Pseudomonas syringae pv tomato DC3000 a model pathogen for probing disease susceptibility and hormonesignaling in plants Annu Rev Phytopathol 51 473-498
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang H Li L Yu Y Mo J Sun L Liu B Li D Song F (2013) Cloning and characterization of two rice long-chain base kinase genesand their function in disease resistance and cell death Mol Biol Rep 40 117-127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L Jia C Liu L Zhang Z Li C Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f splycopersici toxin-induced tomato cell death J Exp Bot 62 5405-5418
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang Y Yang X Zeng H Guo L Yuan J Qiu D (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves diseaseresistance in Arabidopsis thaliana Biotechnol Lett 36 1069-1078
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA (2003) Virulence systems of Pseudomonas syringae pv tomatopromote bacterial speck disease in tomato by targeting the jasmonate signaling pathway Plant J 36 485-499
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zheng XY Spivey NW Zeng W Liu PP Fu ZQ Klessig DF He SY Dong X (2012) Coronatine promotes Pseudomonas syringaevirulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation Cell Host Microbe 11 587-596
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerli L Metraux JP Mauch-Mani B (2001) beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophicfungus Botrytis cinerea Plant Physiol 126 517-523
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
- Parsed Citations
- Article File
- Figure 1
- Figure 2
- Figure 3
- Figure 4
- Figure 5
- Figure 6
- Figure 7
- Figure 8
- Figure 9
- Figure 10
- Figure 11
- Figure 12
- Figure 13
- Figure 14
- Parsed Citations
-