Unité de Bioindustries Passage des Déportés, 2 - 5030 ... · Centre Wallon de Biologie...

CCentre entre WWallon de allon de BBiologieiologie IIndustrielle ndustrielle (CWBI) fond(CWBI) fondéé en 1988en 1988

Université de Liège – Gembloux Agro-Bio Tech

Unité de BioindustriesPassage des Déportés, 2 - 5030 Gembloux – Belgique. Tél+32(0)81 62 2305 (Fax 6142) - www.fsagx.ac.be

De la cellule au produit finiDe la cellule au produit fini

sélection de lasouche

étude de la physiologiemicrobienne

réacteurs

. ... .

. .

.

.

..

technologie de lafermentation

récupération etconditionnement

des cellules ou desmétabolites

4

Fermenteur 2m3

Fermentation

fermenteurs de 2 litres, 20 litres, 250 litres, 500 litres et 2 m3

MatMatéériel disponible au CWBIriel disponible au CWBI

5

Centrifugeuses

Downstream process

centrifugeuse stérilisable, appareil d’ultrafiltation, filtre à plaque, filtre-presse, évaporateur sous vide

Evaporateur sous vide

6

atomiseurs

Conditionnement

Sphéroniseur et extrudeur, mélangeur, atomiseurs, lyophilisateurs, lit fluidisé

lyophilisateur

Université de Liège – Gembloux Agro-Bio Tech – Unité de BioindustriesPassage des Déportés, 2 - 5030 Gembloux – Belgique. Tél+32(0)81 62 2305 (Fax 6142) - www.fsagx.ac.be

Marc [email protected]

Embrapa, September 10, 2010

Molecular basis of biocontrol by Bacillus sp.: Lipopeptides playing in the game

Part I:

Biopesticides, Bacillus and lipopeptides

Why developing (microbial) biopesticides?

• Crop Quality and Yield• protective effect against diseases• Labor and Harvest Flexibility• IPM Compatibility• Resistance Management• Environmental Safety• Residue Management

•Global pesticide market approx. $41 billion (2009)

•Biopesticides market $ 1.6 billion (2009) 15.6% annual growth rate (predicted!)

•Microbial products represent about 30 % of total biopesticide sales

Microbes as biopesticides

Most of the bacterial strains exploited as biopesticides belong to the genera:

Streptomyces, Agrobacterium, Bacillus and Pseudomonas

Bacillus‐based products represent about half of the commercially available bacterial biocontrol agents

(B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus...)

Bacteria as biopesticides

Why is Bacillus among the best candidates for developing biopesticides?

Multifactorial basis!

Efficient producer of antibiotics (more than two dozen)

> antibiosis towards phytopathogens

Efficient colonizer of root systems (soil bacterium)

> compet. for space and nutrients – growth promotion

Bacillus sp. as biopesticide

‐ Controls + Feng

Botrytis‐infected apples

Bs C

Efficient spore former

> persistence ‐ formulation


Efficient enhancer of plant resistance

> Strenghthening of the host

C LP

Beneficial effects of B. amyloliquefaciens strain S499 on tomato in Burundi


And efficient in the field…

Multiple examples of disease control in the litterature

High level of protection (75%) against the disease caused by the endemic Fusarium pathogen

0

1

2

3

4

5

6

7

C 10exp5 10exp6 10exp7 10exp8

Mean diam

eter of n

ecrosis (cm

)

Bacterial inoculum concentration (CFU/ml)

a b c A

B


Growth promotion

Increased fruit yield


What are lipopeptides and why are they interesting?

Biosurfactant… with multiple functions!

LPs from plant‐associated bacteria: a panoply of structures

Pseudomonas, Bacillus, Serratia

Families ‐ Variants ‐ Homologues

Synthesized via NRPS

Surfactins

Heptapeptides cyclic lactone ring

4 structural variantsVarious homologues C12 to C15, linear, iso, anteiso

Iturins

heptapeptides linked to a b‐amino fatty acid

7 structural variants(Bacillomycins, mycosubtilins, iturins A)Homologues C14 to C17

4 structural variantsHomologues from C14 to C18

Fengycins

Lipodecapeptides, internal lactone ring

Bacillus lipopeptides

Colonization, biofilm

Antiviral

Antiprotozoal

Antibacterial

Antioomycete Antifungal

Chelation,solubilization

Motility Virulence,Immunization

Natural functions of LPs

Raaijmakers et al. 2010

involved in biocontrol

Beneficialrhizobacteria

Involvement of surfactins in pellicle/biofilm formation and motility (Hofemeister et al. 2004 Mol. Genet. Genomics; Branda et al. 2001 Proc. Natl. Acad. Sci. U. S. A.)

Role of surfactin in biofilm formation by B. subtilis on Arabidopsis roots (Bais et al. 2004 Plant Physiol.)

FZB42 wtSrf+Fen+

Bac+

AK3 deriv Srf+Fen-

Bac-

CH1 deriv Srf-Fen+

Bac-

CH2 derivSrf-Fen-

Bac+

Involvement in root colonization

Role of massetolide in colonization of tomato roots (Tran et al. 2007 New Phytol.)

‐ Spreading (reduce surface tension, wettability agents)‐ Attachement (hydrophobic surfaces, plant tissues)‐ Biofilm developement

Pathogen

Involvement in direct antagonism

- LP + LP

Bacterial cell/fungal spore lysis, inhibition of hyphal developement

Soil‐borne diseases: iturins involved in the control of R. solani (Asaka, Shoda 1996 Appl Environ Microbiol) and P. aphanidermatum on tomato (Leclère et al. 2005 Appl. Environ. Microbiol.)

Phyllosphere diseases: surfactins for reduction of the infection caused by P. syringae on Arabidopsis. Iturins and fengycins in the antagonism toward Podosphaera fuscainfecting melon leaves

Post harvest diseases: fengycins in gray mold disease (Botrytis cinerea) reduction on apple fruits (Ongena et al. 2005 Appl. Microbiol. Biotechnol.)

Detergent, pore forming activity

- LP + LP

Pathogenrestriction

Host plant immunization

C LP

Involvement of LPs in ISR elicitation

● Disease reduction on leaves upon root treatment with purified LPs

● Disease reduction on leaves upon root treatment with mutants

● No migration of inducing agent from roots to the infected leaves

● Efficient production of the inducing agent in the rhizosphere

● Protection associated with defence responses at the molecular level

Specific biocontrol‐related activities for the three Bacillus LP families

powerful biosurfactants

Antiviral

Antibacterial

low fungitoxicity

Strongly antifungal (yeast and fungi)

low antibacterial

no antiviral activities

Surfactins Iturins

strongly fungitoxic (filamentous fungi )

Fengycins

Root colonization (biofilm formation, surface motility)

Direct antagonism toward phytopathogens

Involvement in plant systemic resistance elicitation

Still a very small part of the global market for Bacillus-based biocontrol products…

Why are microbial biopesticides not so successful ?

Limitations in efficacy – Inconsistency !

Need for a better understanding of what happens in the phytosphere regarding secretion of bioactive compounds

‐ Auxiliary microflora‐ Root zone‐ Soil type, heterogeneity‐ pH, T, pO2

‐ Plant species, cultivar, developmental stage

Selection of specific microbial populations by the plant through :

‐ Release of soluble carbon compounds and other rhizodeposition‐ pH and redox‐modulating factors‐ Release of complexing agents (siderophores, phenols, carboxylates)‐ Release of antimicrobials (antibiotics, quorum sensing inhibitors)‐ Exudation of specific stimulatory compounds‐ Shaping specific habitat conditions

Rhizosphere factors influencing microbes

Are LPs actually secreted upon growth of Bacillus on plant roots?

What’s the influence of rhizosphere‐specific parameters on LP production rate?

Is the LP signature influenced by the host plant species ?

Not only microbial communities but also expression of biocontrol traits may be influenced by the host plant type and environmental factors

Part II:

In situ production of Bacillus lipopeptides

1. LP production on various host plants

B. subtilis S499: a natural strain

Efficient producer of the three LP families srf, fen and itu

recalcitrant to transformation

Determination of the LP pattern produced in the rhizospheres(quantitative, qualitative)

Differential expression of srf, fen and myc genes

Development of extract./LC‐DAD‐ESI‐MS methods

LP production in the rhizosphere of hydroponic plants (non sterile)

LC‐MSExtraction

Populations on most plants > 105 cfu/g root FW but estimated

The three LP families are differentially produced in the rhizosphere

LP production in the rhizosphere of in vitro‐grown plants (sterile)

Colonization (cfu/g root FW, 2 weeks p.i.)

corn lettuce bean tomato cucumber soy

2,6. 106 2,8. 107 1,8. 107 2,3. 107 nd nd

Imaging TOF‐MS

Transfer on silicium plate

LP analysis

Control

Bacillus‐colonized root

Srf

Itu

Fen

Inner MS cameraScanned zone

Fingerprint on silicium plate

Srf

Itu

Fen

Larger root section

LP production in exudates

Exudates collected from hydroponic and non‐treated plants (4 weeks)

Root exudates seem to be more conducive for itu and srf synthesis

srf

itu

fen

LP production in individual C sources

Liquid cultures to test substrates individually

Exudates recomposed on the basis of substrates typically found in tomato as described by Kamilova et al., 2006

Higher srf production in the presence of organic acids

C14 and C15 are the forms mainly produced also in the presence of root exsudates

cLPs synthesis also influenced qualitatively…

Long chain homologues seem to be favored

Generally:

LP signature may be strongly influenced by the host plant species

‐> Better to use of the Bacillus strain for biocontrol on some specific crops?

In summary…

Quantitatively:

In half of the plants tested so far, srf is seemingly the main LP secreted by S499

Fen are very poorly detected in the rhizosphere. Does it reflect reality?

300 ng/g root FW corresponds to ~ 0.12 µM in the hydroponic medium. Is it sufficient? LPs are active at 5‐30 µM concentrations

Qualitatively:

Long chain homologues seem to be favored (tomato and corn)In some instances, long chain LP homologues demonstrated to be more active

2. More insights about surfactin synthesis in the rhizosphere

Part II:

In situ production of Bacillus lipopeptides

1. LP production on various host plants

Combination of two approaches:

Ex vivo: batch cultures, bioreactor/chemostat

In vivo: mass spectrometry, reporter genes lacZ

BGS3 derivative < BC25: lacZ reporter system under the control of the psrf gene

thrCpsrf

lacZ thrC

B. subtilis BGS3: a « laboratory » strain

Expression of srfA genes in the rhizosphere

Higher surfactin gene expression during the period where BGS3 cells maintain a stable population

Confirmation of relatively high srfA expression at low growth rate

Influence of µ tested in chemostat cultures

Growth rate

Surfactin production in the rhizosphere

Collection of roots and nutrient solution

C18 extract. of NS

MeOH/Triton extract of root surface

C18 extract.

320 ± 80 µg surfactin / 108 cells= approx. 1.8 µM in the plant growthmedium

LC-MS

+

Surfactin recovery from the rhizosphere in the range of concentrations necessary for triggering plant resistance or defense reactions (2-5 µM)

Gene expression Surfactin productionβ‐gal U µg/108 cells

Optimized medium 53 ± 11 470 ± 35

Natural exudates 17 ± 3 140 ± 40

Recomposed exudates 25 ± 1.7 340 ± 75

srfA gene expression and lipopeptide secretion into the medium also effective upon growth of BGS3 in tomato root exudates

Surfactin synthesis in root exsudates

Exudates collected from non‐bacterized tomato plantlets after 23‐25 days of growth in nutrient solution under sterile conditions (suppl. (NH4)2SO4 )

Exudates recomposed on the basis of substrates typically found in tomato exudates as described by Kamilova et al., 2006

Liquid cultures to test substrates individually (flasks)

Effect of carbon sources on surfactin synthesis

Biomass production

Higher cell concentration in sugars and a.a. but efficient growth on most substrates except Ara

srfA gene expression


Market effect of Ara ?Higher expression in the presence of a.a. and some organic acids

Surfactin production

Higher production in organic and amino acids

C14 and C15 mainly produced are also the more active forms


No significant effect of cell immobilization/development on solid support/roots

Similar trends in surfactin gene expression and production on gelified media

Other clues…

Higher surfactin production in the presence of Fum, Mal, Cit, Asp, Glu

Higher gene expression in the presence of organic acids and a.a.

Culture conditionsBiomass

(cells x 108/ml)

srfA expression

(b‐gal /108 cells)

Surfactin prod.

(mg/108 cells)

Bioreactor, 1 vvm, 300 rpm 6.1 54 550

Aerated flask 5.6±0.3 47±11 660±70

Sealed flask + nitrogen flushed 3.3±0.7 18±4 480±40

Limited effect of low aeration rate on surfactin synthesis

Other clues…

Low oxygen availability in soil not detrimental to surfactin production

Other clues…

Effect of Temperature

Effect of pH

Rhizosphere conditions conducive to surfactin secretion by B. subtilis with regards to: ‐ exudates

‐ growth rate‐microcolonies‐ oxygen status

In summary…

The surfactin pattern may also be influenced by‐ the nutritional basis imposed by the host plant‐ the immobilization (not shown)‐ oxygen availability (not shown)

Need for a better understanding of the mode of action of the bioactive compounds

Why are microbial biopesticides not so successful ?

Limitations in efficacy – Inconsistency !

Part III:

Bacillus lipopeptides as elicitors of resistance in the host plant

1. Demonstration for a role of LP in triggering systemic resistance

Primed state

Pathogen

Infection

Colonization

PGPR

Induced resistance state

Pathogen restriction

Rhizobacteria‐induced systemic resistance (ISR) in plants

Disease reduction on leaves upon root treatment with purified LPs

Demonstration of LP involvement in ISR stimulation

ISR activitytomato bean cucumber tobacco

B. subtilis strain S499 ++* ++* ++* ++* Surfactin (5‐10 µM) ++* ++* ‐ ++

P. fluorescens SS10 ++* n.t. n.t. n.t.Massetolide (22‐44 μM) +* n.t. n.t. n.t.

++, 25‐40% disease reduction*, statistically different from disease controln.t., not tested

Disease reduction on leaves upon root treatment with mutants


ISR activitytomato bean cucumber tobacco

B. subtilis wt 168 ‐ ‐ n.t. n.t.Surfactin overproducer ++* ++* n.t. n.t.

P. fluorescens SS10 ++* n.t. n.t. n.t.Massetolide suppressed ‐ n.t. n.t. n.t.

++, 25‐40% disease reduction*, statistically different from disease controln.t., not tested

No migration of inducing agent from roots to the infected leaves


OK

Production of the inducing agent in the rhizosphere

Macroscopic protection associated with defence responses at the molecular level


Stimulation of early defence events (oxidative burst) in roots of plants treated with surfactin

Srf

Ctrl

Time in min

0 30LOCAL

Not the « SAR‐type » ISR

SIGNALLING1 2 3

1: BTH treated plants; lanes 2: control plants; 3: Bac. S499‐treated plants

> No (free) SA accumulation and no PR protein expression in plants induced by surfactin producers

> ISR induced by massetolide also in nahG tomato plants no longer able to accumulate SA

PR1

Nucleus

H2O2NADPH oxydase

2 O2 2 O2.-

2 NADPH2 NADP + 2 H+

SOD

H2O2

H+

ATP ADP+Pi

NO3-

Cl-K+

H+

Ion fluxespH

Ca2+

Unsaturated fatty acids

Kinases Defense gene activation

PL A2PL C

PALLOX

Phenoliccompounds

Octadecanoidpathway

?

Oxidative burst

EARLY EVENTS INDUCED IN TOBACCO CELLS

Macroscopic protection associated with defence responses at the molecular level


Stimulation of key enzyme activities (LOX, LHP) of the oxylipin pathway within 48 h.p.i. (new LOX gene isoform) in leaf tissues

possible accumulation of antifungal volatiles and/or signalling compounds (JA)

0 48 96 0 48 96 MeJA

Control Bs2500

Lox F

C C CBs Bs Bs0 48 96 hpi

> Accumulation of antifungal compounds (phytoalexins) in leaves of plants treated with surfactin overproducer isolate

SYSTEMIC

Surfactins stimulate ISR in various plants, fengycins are also active albeit to a lower extent, but iturins are not

Fengycins and surfactins (less active) induce phenolic accumulation in potato tuber cells but iturin do not.

In tobacco cells, surfactins trigger a whole set of defence‐related early events while fengycins only stimulate later enzyme activities and iturins are not active.

Differential activity of LP families on plants

From Bacillus:

Surf

Itu

Feng

2. How do LPs work at eliciting plant cells?

Part III:

Bacillus lipopeptides as elicitors of resistance in the host plant

1. Demonstration for a role of LPs in triggering systemic resistance

More insights into the molecular interaction of cLPs with plant cells

elicitorsreceptors bilayer perturbation?

Elicitor perception

Rhizobacteria

Early events

FlagellaLipopolysaccharidesBiosurfactants

lipopeptidesrhamnolipids

N‐acyl‐L‐homoserine lactoneN‐alkylated benzylamineSiderophores

1. Pseudobactins SA and SA‐containing siderophores.

Antibiotics1. 2,4‐Diacetylphloroglucinol2. Pyocyanin

VolatilesExopolysaccharides

ISR elicitors from rhizobacteria

Receptor (also PAMP)Low‐affinity receptor (also PAMP)??

?????? (iron stress)

??

????

Defense‐related events stimulated by LPs in cultured plant cells (Tobacco)

Nucleus

H2O2NADPH oxydase

2 O2 2 O2.-

2 NADPH2 NADP + 2 H+

SOD

H2O2

H+

ATP ADP+Pi

NO3-

Cl-K+

H+

Ion fluxespH

Ca2+

Unsaturated fatty acids

Kinases Defense gene activation

PL A2PL C

PALLOX

Phenoliccompounds

Octadecanoidpathway

?

Oxidative burst

Early and later defense events elicited by surfactin in plant cells

Differential activities of the 3 families (fengycin only induce later

response, iturin not active)

> role of structural features in the peptide?

Similarities with the early responses to PAMPs

Mechanism of surfactin perception

Strong interaction with lipid bilayers in model systems

Biocidal activity relying on pore formation within the envelop of the target (virus, bacteria)

Specific receptors? Interaction with plasma membrane ?

0

20

40

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80

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140

160

180

200

Mix C12 C13 C14 ni C14 nn C14 ni,m C14 ni,l C14 ni,lm C15

Activity (%

)

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Mix C12 C13 C13 Val7 C13 Leu4 C14 C14 Leu4 C14 Ile C15 C15 Val7 C15 Leu4 C15 Ile

Activity (%

)

■ Structure/activity: comparison of multiple homologues and variants

Specific aspects in the interaction between surfactin and plant cells

Chemical synthesis Metabolic engineering

LinearizedMethylated

Natural co-production

C12, C13, C14i+n, C15 Amino-acid substitutions(Leu<>Val<>Ile)

■ Structure/activity: the more lipophilic the more active, crucial role for the amphiphilic character, the neo‐synthesized peptide forms are the more active


■ Structure/activity: no crucial role for a.a. sequence, the more lipophilic the more active, crucial role for the amphiphilic character

■ No competitive effect between homologues




0

0,2

0,4

0,6

0,8

1

1,2

0 2 4 6 8 10 12 14 16 18 20

ΔpH

Surfactin concentration (µM)

C

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14

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18

0 5 10 15 20

Rel

ativ

e H

202

accu

mul

atio

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Surfactin concentration (µM)

B

■Micromolar concentrations required (EC50 ~ 2.5 µM) for activity





■ No secondary oxidative burst associated with HR and very limited cell death






■ No refractory state phenomenon

S C14, 10 µMS C14, 10 µM







■ No refractory state phenomenon

> Talk against the presence of a high affinity/receptor‐based surfactin recognition system in plants

■ Surfactin fully insert into plasma membrane fraction



■ Higher affinity for membrane of long‐ chain surfactins, significant decrease in affinity for methylated and/or linear surfactins



■ Higher insertion rate in membrane for long‐ chain surfactins, significant decrease for methylated and/or linear surfactins

■ No significant change in insertion rate of C14 surfactins in heat‐treated or protease‐treated cells/protoplasts (not shown)





■ Data from Iso Thermal Calorimetry using model vesicles


Surfactin activity on eucaryotic cells:not or poorly active on fungi> 15 µM (not toxic) on mammalian cells, within hours> 2.5 µM (not toxic) on tobacco cells, within minutes

Affinity related to phospholipid and/or sterol content of plasma membrane?

> Specific targetting in rafts?

Martin et al. (2005) Trends Plant Sci.

Surfactin interacts spontaneously with model vesicles, in an endothermic and entropy‐driven process > Confirm strong hydrophobic and dose‐dependent interactions (no signal < 2µM, similar as biological activity on tobacco cells)

Lipid composition of vesicles K (mM‐1)POPC 1mM 39,5±4,1PLPC 1mM 43,1±1,3PLPC/Stigmasterol 9/1 1mM 39,2±3,8PLPC/Stigmasterol 3/1 1mM 27,4±3,4PLPC/Ergosterol 3/1 1mM * 34,0±0,8DPPC/PLPC 3/1 1mM 61,1±5,3DPPC/POPC 3/1 1mM * 85,4±8,5* Result from one measurement

Strong difference in partitioning constants between C13 and 14 homologues as well as between surfactin and fengycins and iturins which interacted spontaneously (ΔG<0) but in an exothermic manner with vesicles by contrary to surfactins

Lipid composition of vesicles K (mM-1)Surfactin C13 8,3Surfactin C14 30,9Surfactins mix 54Fengycins 9,4Iturins 31,8

Close relationship between the physical structure of the lipid bilayer and the association of surfactin with the bilayer

Lipid composition of vesicles Lipid phases KPOPC/PSM 0.1/0.9 GelPOPC LdPOPC/Chol 0.45/0.55 Lo POPC/PSM/Chol 0.5/0.25/0.25 Ld + Lo (raft)POPC/PSM/Chol 0.2/0.5/0.3 Lo + GelPOPC /PSM 0.6/0.4 Ld + GelPOPC/PSM/Chol 0.4/0.5/0.1 Ld + Lo + Gel




■ Data from Iso Thermal Calorimetry using model vesicles


Talk in favor of a role as “sensor”of the plasma membrane with surfactin‐induced disturbance or rearrangement, lauching defense signalling cascade

Favour the ecological fitness of the producing isolates in the phytosphere:

> Niche colonization (motility, biofilm)> Competitive interactions with the other organisms

More than just antibiotics!

> Immuno‐stimulation in plants

May impact on various cell processes without causingany detrimental leakage in membrane

‐ Cytotoxicity /antagonism‐ Inhibition of pathogen attachement/biofilm ‐ Interference with fungal toxin synthesis (fumonisin, aflatoxin)‐ Signalling in molecular dialogue with predators

Molecular pneumatic drills

and

In conclusion

What factors drive the perception at the membrane level?

Cannot conclusively rule out the involvement of low‐affinity proteic receptors

‐ Testing other structural variants on tobacco cells and in ITC‐ Testing reactivity of cells from other plants with different phospholipids‐ Testing binding of radiolabelled surfactin on proteic fraction of plant membrane

Surfactin among the rare Bacillusmetabolites identified so far as plant immuno‐stimulators (2,3‐butandiol)

Shen et al. 2009

Competitive advantage of an efficient production of surfactin, fengycin and iturin with specific roles and targets (synergistic effects)

May explain why some Bacillus or Pseudomonas strains are efficient and others not

Rapid method of screening for the selection of useful strains that co‐produce the broadest panoply of LP families should display a multi‐faceted biological control

In the context of biocontrol

Thank you…

Financial support from FRS‐F.N.R.S, F.R.I.A. and Walloon Region in Belgium.

For more information:

Ongena et al. 2007 Environ. Microbiol. 9, 1084Adam 2008 PhD thesis, Univ. LiègeAdam et al. 2008 BMC Plant Biol. 8, 113Jourdan et al. 2009 Mol. Plant Microbe Interact. 22, 456Nihorimbere et al. 2009 Environ. Microbiol. Rep. 1, 124

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