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Evolutionary Conserved Function of Barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in Providing Wax Signals for Germination of Powdery Mildew Fungi 1[C][W] Denise Weidenbach 2 , Marcus Jansen 2 , Rochus B. Franke, Goetz Hensel, Wiebke Weissgerber, Sylvia Ulferts, Irina Jansen, Lukas Schreiber, Viktor Korzun, Rolf Pontzen, Jochen Kumlehn, Klaus Pillen, and Ulrich Schaffrath* Department of Plant Physiology, Rheinisch-Westfaelische Technische Hochschule Aachen University, D52056 Aachen, Germany (D.W., M.J., S.U., I.J., U.S.); Institute of Biosciences and Geosciences: Plant Sciences, Juelich Plant Phenotyping Centre, Forschungszentrum Jülich GmbH, 52425 Juelich, Germany (M.J.); Ecophysiology of Plants, Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany (R.B.F., L.S.); Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Stadt Seeland/OT Gatersleben, Germany (G.H., J.K.); Institute of Agricultural and Nutritional Sciences, Chair of Plant Breeding, Martin Luther University Halle-Wittenberg, 06120 Halle/Saale, Germany (W.W., K.P.); Cereals Biotechnology, KWS LOCHOW GMBH, 37574 Einbeck, Germany (V.K.); and Formulation Technology, Bayer CropScience AG, 40789 Manheim, Germany (R.P.) For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identi ed to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has signicantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our ndings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago. Aerial parts of plants are usually covered with a cuticle. This interface between an organism and the environment developed in ancient times as a prerequisite for pioneer- ing plants when leaving their water home and occupying dry land (Bargel et al., 2004). Other than its unquestionably important function in protecting plants from desiccation, cuticles also represent the outermost barrier that shelters plants against pathogen and pest attacks. The cuticle has a water-repellent effect, which from the physical point of view, enhances slipperiness and thereby, impedes the ability of nonspecialized microbes to get in touch with a potential host (Howe and Schaller, 2008). Thus, in the course of evolution, pathogens had to develop strategies to cope with the cuticle barrier in order to become a successful invader. During this process, however, some pathogens might have started to utilize cuticle-derived signals in a more sophisticated fashion: as a clue to initiate accelerated germination only in the presence of potential plant hosts (Lapin and Van den Ackerveken, 2013). Generally, the cuticle is composed of the cutin polymer matrix and cuticular wax. Cuticular wax is embedded within the cutin polymer (intracuticular wax) and on the outer surface (epicuticular wax). Epicuticular wax often (but not in all species) forms three-dimensional crystallites like, for example, in barley (Hordeum vulgare; Jetter et al., 1 This work was supported by the Bundesministerium für Bildung und Forschung (Funding Activity Plant Biotechnology for the Future, PLANT 2030 within project BarleyFortress to D.W.). 2 These authors contributed equally to this work. * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Ulrich Schaffrath ([email protected]). [C] Some gures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.114.246348 Plant Physiology Ò , November 2014, Vol. 166, pp. 16211633, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved. 1621 www.plantphysiol.org on July 17, 2020 - Published by Downloaded from Copyright © 2014 American Society of Plant Biologists. All rights reserved.

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Evolutionary Conserved Function of Barley andArabidopsis 3-KETOACYL-CoA SYNTHASES inProviding Wax Signals for Germination ofPowdery Mildew Fungi1[C][W]

Denise Weidenbach2, Marcus Jansen2, Rochus B. Franke, Goetz Hensel, Wiebke Weissgerber,Sylvia Ulferts, Irina Jansen, Lukas Schreiber, Viktor Korzun, Rolf Pontzen, Jochen Kumlehn,Klaus Pillen, and Ulrich Schaffrath*

Department of Plant Physiology, Rheinisch-Westfaelische Technische Hochschule Aachen University, D–52056Aachen, Germany (D.W., M.J., S.U., I.J., U.S.); Institute of Biosciences and Geosciences: Plant Sciences, JuelichPlant Phenotyping Centre, Forschungszentrum Jülich GmbH, 52425 Juelich, Germany (M.J.); Ecophysiology ofPlants, Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany (R.B.F., L.S.);Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Stadt Seeland/OTGatersleben, Germany (G.H., J.K.); Institute of Agricultural and Nutritional Sciences, Chair of Plant Breeding,Martin Luther University Halle-Wittenberg, 06120 Halle/Saale, Germany (W.W., K.P.); Cereals Biotechnology,KWS LOCHOW GMBH, 37574 Einbeck, Germany (V.K.); and Formulation Technology, Bayer CropScience AG,40789 Manheim, Germany (R.P.)

For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matterof vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible.We investigateda barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildewfungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screenand identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required forsynthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly feweraliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcamegermination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigationof Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAXPRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powderymildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly relatedpowdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before themonocot-dicot split took place approximately 150 million years ago.

Aerial parts of plants are usually coveredwith a cuticle.This interface between an organism and the environmentdeveloped in ancient times as a prerequisite for pioneer-ing plants when leaving their water home and occupyingdry land (Bargel et al., 2004). Other than its unquestionablyimportant function in protecting plants from desiccation,

cuticles also represent the outermost barrier that sheltersplants against pathogen and pest attacks. The cuticle hasa water-repellent effect, which from the physical point ofview, enhances slipperiness and thereby, impedes theability of nonspecialized microbes to get in touch with apotential host (Howe and Schaller, 2008). Thus, in thecourse of evolution, pathogens had to develop strategiesto cope with the cuticle barrier in order to become asuccessful invader. During this process, however, somepathogens might have started to utilize cuticle-derivedsignals in a more sophisticated fashion: as a clue to initiateaccelerated germination only in the presence of potentialplant hosts (Lapin and Van den Ackerveken, 2013).

Generally, the cuticle is composed of the cutin polymermatrix and cuticular wax. Cuticular wax is embeddedwithin the cutin polymer (intracuticular wax) and on theouter surface (epicuticular wax). Epicuticular wax often(but not in all species) forms three-dimensional crystalliteslike, for example, in barley (Hordeum vulgare; Jetter et al.,

1 This work was supported by the Bundesministerium für Bildungund Forschung (Funding Activity Plant Biotechnology for the Future,PLANT 2030 within project BarleyFortress to D.W.).

2 These authors contributed equally to this work.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ulrich Schaffrath ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.114.246348

Plant Physiology�, November 2014, Vol. 166, pp. 1621–1633, www.plantphysiol.org � 2014 American Society of Plant Biologists. All Rights Reserved. 1621 www.plantphysiol.orgon July 17, 2020 - Published by Downloaded from

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2000; Kunst and Samuels, 2003; Bargel et al., 2004). Cutinis a covalently cross-linked polymer made of saturatedC-16-hydroxy and partially unsaturated C-18-hydroxy andC-18-epoxy fatty acids (Bargel et al., 2004). Cuticular waxes,by contrast, are complex organic solvent-extractable mix-tures of monomeric C-20 to C-60 aliphatics that may in-clude triterpenoids, phenylpropanoids, and flavonoids(Samuels et al., 2008). The composition of intracellular andextracellular waxes is extremely variable and may differbetween plant species, organs within a given species,and also, developmental stages of plant organs (Post-Beittenmiller, 1996; Bargel et al., 2004). Wax biosynthesisstarts in leucoplasts, the photosynthetically inactive plastidsof the epidermis, with de novo synthesis of C-16 and C-18fatty acids. These are subsequently elongated andmodifiedin the endoplasmic reticulum to, at first, very long-chainfatty acids (VLCFAs; C-20 to C-34) and then, alcohols,aldehydes, esters, alkanes, and ketones (Samuels et al.,2008). The plastidial fatty acid synthesis and the extensionto VLCFAs are carried out by multienzyme complexestermed fatty acid synthases and fatty acid elongases(FAEs), respectively; both consist of four dissociableenzymes catalyzing consecutive enzymatic reactions(Samuels et al., 2008). For elongation, C-2 units derivedfrom malonyl-CoA are added to fatty acids or VLCFAsby the FAE complex, involving condensation, reduc-tion, dehydration, and a second reduction step. Severalcycles are needed to yield chain lengths of, for example,C-24 to C-34. Each of those cycles seems to be carried outby different FAE complexes, which are distinct by indi-vidual b-KETOACYL-CoA SYNTHASEs (KCSs), becausetheir specificity is in the KCS condensing enzyme (Samuelset al., 2008; Chen et al., 2011; Haslam and Kunst, 2013).Therefore, it is not surprising that a large family of KCSsexists (e.g. 21 KCS-like sequences have been identified inArabidopsis [Arabidopsis thaliana]; Kim et al., 2013). How-ever, the function in elongation of epicuticular wax VLCFAswas experimentally verified so far for only two of theseenzymes (AtKCS1 and Arabidopsis ECERIFERUM6[AtCER6]/Arabidopsis REQUIRED FOR CUTICULARWAX PRODUCTION1 [AtCUT1]/AtKCS6; Millar et al.,1999; Todd et al., 1999; Fiebig et al., 2000).

Our current understanding of the wax biosyntheticpathway and the precise function of particular enzymesis still limited, although genetic as well as biochemicalapproaches have been used widely. Thereby, forwardgenetic screens profited from the easy to score visualphenotype of mutants with little or no wax coating onaerial plant organs, which appear glossy or glaucous. Inbarley, these mutants are termed eceriferum (Latin: cerameans wax and ferre means to bear), and cer is used asthe symbol for the respective gene loci (Lundqvist andvonWettstein, 1962). The Arabidopsis community followedthis terminology, and today, 85-cer and 22-cer loci havebeen identified in barley and Arabidopsis, respectively(Kunst and Samuels, 2003). However, because thesemutants are affected in their total wax load rather thandifferences among wax components, it is unlikely thatthey cover the whole wax biosynthetic pathway (Post-Beittenmiller, 1998). In Arabidopsis, this gap was closed

by reverse genetic approaches, which enabled the identi-fication of additional genes involved in cuticular wax bio-synthesis (Kunst and Samuels, 2003). Sequence homologiesto some of these genes were used to clone candidate genesof barley (Richardson et al., 2007). However, because for-ward and reverse genetic data are missing, gene-functionrelationships in barley are still speculative.

Airborne plant pathogens, regardless of whether theyare dispersed by water or wind, have to cope with theproblem of adhesion to the waxy surface of their hostplants. In the case of water-disseminated pathogens, suchas Magnaporthe oryzae or Colletotrichum graminicola, this isachieved by release of glue, whereas for wind-disseminatedpathogens, like powdery mildews, the initial contact can besecured by hydrophobic interactions between the con-idiospore and the leaf surface (Epstein and Nicholson,2006). Nevertheless, conidial extracellular material mightalso help in this case to attach a conidiospore to the plantsurface (Wright et al., 2002). After attachment, pathogenshave to overcome the cuticle and the epidermal cell wallbarriers to reach the nutrient pool of the plant interior.This process is referred to as penetration, and it mightinvolve the generation of high turgor pressure in dedi-cated cells (so-called appressoria) to mechanically breachthe cell wall, which is eventually supported by the ac-tivity of hydrolytic enzymes (Tucker and Talbot, 2001).For example, in Blumeria graminis f. sp. hordei (Bgh; the barleypowdery mildew fungus), a combination of turgor-drivenand enzymatic-supported penetration has been suggested(Pryce-Jones et al., 1999). Generally, powderymildews arehighly specialized biotrophic pathogens infecting and re-producing only on living tissue of a limited range of plantspecies (Both and Spanu, 2004). The disease cycle of Bgh,which uses barley as the sole host, starts with the attach-ment of a conidiospore to the leaf surface and the formationof a primary germ tube (Carver et al., 1995). In addition, anappressorial germ tube is formed, giving rise at its tip to anappressorium from which the fungus penetrates the un-derlying tissue. In the case of success, the fungus invadesan epidermal cell and forms his feeding organ, the so-calledhaustorium, while leaving the plasma membrane intact.Thereafter, the pathogen develops secondary hyphae onthe leaf surface and continues to penetrate neighboringepidermal cells. The fungus completes its lifecycle bybuilding conidial mother cells, and novel conidiosporesemerge (Eichmann and Hückelhoven, 2008). Right afterinitial contact, Bgh conidiospores prepare an infection courtat the interface to their host, and Epstein and Nicholson(2006) speculated that, at this space of tight adherence, theconcentration of lytic enzymes, such as cutinases (Pascholatiet al., 1992), effectively could be maintained at higher levels.This enzymatic activitymay lead to the release ofmonomericor oligomeric degradation products (e.g. cutin monomers),which can act as damage-associated molecular patternsand trigger defense responses (Schweizer et al., 1996;Tucker and Talbot, 2001). The involvement of epicutic-ular wax components in defense was shown in a recentstudy, in which silencing of a cytochrome P450 gene,involved in the generation of VLCFA derivatives such assecondary alcohols and ketones, diminished penetration

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resistance of barley against M. oryzae (Delventhal et al.,2014). By contrast, cutin monomers may also contributeto disease susceptibility, because, for example, in Bgh andUstilago maydis, they play a role in appressorial germ tubeand appressorium formation, respectively (Francis et al.,1996; Mendoza-Mendoza et al., 2009). For the wheatpowdery mildew fungus B. graminis f. sp. tritici (Bgt), itwas shown that the expression of a secreted lipase in-volved in the release of epicuticular wax componentsfrom infected wheat (Triticum aestivum) leaves was im-portant for fungal adhesion and development (Feng et al.,2009). Additional fatty acid components supportingpathogen development rather than acting as damage-associated molecular patterns are long-chain and verylong-chain alcohols and aldehydes from the epicuticularwax layer, which play a crucial role in spore germination ofBgh (Hansjakob et al., 2010), Puccinia emaculata, Phakopsorapachyrhizi, andColletotrichum trifolii (Uppalapati et al., 2012).For the latter three pathogens, a gene involved in thegeneration of respective wax compounds was cloned fromMedicago truncatula and turned out to be a transcriptionfactor affecting wax biosynthetic genes. For Bgh, however,no wax biosynthetic pathway gene involved in spore ger-mination has been cloned so far from its intrinsic barleyhost. Thus, evidence for the involvement of VLCFA de-rivatives in Bgh germination comes from experiments withnonhost wax mutant plants, such as maize (Zea mays) orArabidopsis, and chemical complementation with partic-ular VLCFA derivatives (Hansjakob et al., 2010; Weis et al.,2014). An important issue that, therefore, could not betouched so far is the question of whether the reduced Bghconidial germination rate affects disease severity.Here, we close this long-lasting gap by identifying the

barleyKCS geneHvKCS6 as being required for germinationof Bgh conidiospores on its host. We verified (by genomiccomplementation of the respective mutant and gas chro-matography [GC] -mass spectrometry [MS] analysis of waxcomponents) that this gene encodes a condensing enzymethat is part of the fatty acid elongation complex and hasa presumed specificity for elongation of C-24 to C-26VLCFAs. Comparative analyses with Arabidopsis re-vealed a conserved function of the orthologous gene inproviding essential signals for germination of conidiosporesfrom different powdery mildew species. Using compatiblehost-pathogen combinations, we showed that, on barleyand Arabidopsis wax mutant plants, a reduced germina-tion rate of powderymildew conidiospores finally resultedin less frequently formed disease symptoms, thus openingthe road, to our knowledge, to a new breeding trait.

RESULTS

Germination of Bgh Conidiospores Is Compromised onBarley Mutant Enhanced Magnaporthe Resistance Gene1

The barley mutant enhanced Magnaporthe resistancegene1 (emr1), generated in our laboratory, was identifiedin a suppressor screen for restoration of resistance againstM. oryzae in the hypersusceptible genetic background

mildew resistance locus O allele5 (Ingridmlo5; Jansen et al.,2007). Based on visual scoring of disease symptoms, nophenotypic response was observed against other im-portant barley leaf pathogens (e.g. powdery mildew, netblotch [Drechslera teres], leaf scald [Rhynchosporium secalis],or rust [Puccinia hordei]; Jansen and Schaffrath, 2009). Forpowdery mildew, however, the presence of the strongresistance allele mlo5 could have masked potential effects.Therefore, we reevaluated the interaction between Bghand emr1 mutant plants using a microscopic assay andanalyzed the formation of initial infection structures asdepicted in Supplemental Figure S1. In this experiment,the percentage of conidiospores that did not germinateon leaves of emr1 mutant plants was almost 2 times ashigh (33%) compared with those on leaves of its ancestorIngridmlo5 (18%; Fig. 1A). Germinated conidiosporesgave rise to mature appressoria at a similar rate on bothgenotypes (i.e. on Ingridmlo5 plants, 82% germinationand 72% appressoria; on emr1 mutant plants, 67% ger-mination and 55% appressoria). Thus, apart from com-promised germination, no additional differences werefound in the prepenetration process of Bgh. These resultsshow that the emr1 mutant exhibits two different pheno-types, one of which is the enhanced resistance against M.oryzae and the other is a reduction in the germinationfrequency of Bgh conidiospores. The following experi-ments were designed to answer the question of whetherboth phenotypes are conferred by the same mutationand identify the underlying gene(s).

emr1 Is Depleted in Leaf Surface Waxes

During inoculation, a higher capacity for water retentionwas observed on leaves of the emr1mutant comparedwithother barley cultivars (Fig. 2A). This observation, togetherwith a glossy appearance of emr1 leaves, was reminiscentof barley eceriferum (cer) mutant plants with altered leafwax coating (Post-Beittenmiller, 1996). We applied scan-ning electron microscopy (SEM) to further investigate thisphenomenon and found a strong reduction of wax crystalson the leaf surfaces of emr1 mutants compared with thoseof Ingridmlo5 plants (Fig. 2B). Taking a closer look, it ap-pears as if particular smooth, platelet-like structures of waxcrystals are absent on mutant leaves. Applying GC-MS,the difference between Ingridmlo5 and emr1mutant plantsin total content of cuticular leaf wax could be quantified to10 and 2.4 mg cm22, respectively (Fig. 2C).

Based on the SEM pictures, we speculated that emr1mutants lack particular components of the epicuticularleaf wax. We followed this thought in more depth bydetailed GC-MS analysis of individual wax components.Strikingly, we determined that the most abundant com-ponent among barley cuticular leaf waxes, the C-26 alco-hol (hexacosanol), was reduced by 90% on primary leavesof emr1 mutant plants compared with IngridMLO andIngridmlo5 plants, respectively (Fig. 3; Supplemental Fig.S2). Similar to the decrease in hexacosanol content, theamount of the C-26 aldehyde (hexacosanal) dropped from0.3 mg cm22 on Ingridmlo5 plants to 0.007 mg cm22 on emr1

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Barley HvKCS6 Controls Bgh Germination

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plants. Concomitant with the reduction in wax compo-nents with C-26 chain lengths, an increase of the C-24alcohol (tetracosanol) was measured on emr1 (Fig. 3),suggesting a block in elongation of VLCFAs from C-24 toC-26 chain lengths in emr1 plants. This hypothesis issupported by the observation that C-26-based componentswere also absent in wax esters of emr1 mutant plants(Supplemental Fig. S2). Interestingly, emr1 plants seem tocompensate for the lack of C26 constituents in esters byforming novel alcohol-acid combinations to keep the totalchain length (as the sum of alcohol and acid). For example,in Ingridmlo5 plants, the ester with a chain length of C-46is built from the C-26 alcohol and the C-20 acid, whereas inemr1 mutant plants, the C-46 ester is made from the C-24alcohol and the C-22 acid or the C-22 alcohol and the C-24acid (Supplemental Fig. S2).

Mapping and In Silico Identification of the GeneResponsible for the Wax Phenotype

On the basis of the observation that emr1 leaves aredepleted in cuticular VLCFA derivatives and in light ofthe works published by Hansjakob et al. (2010, 2011),which show that the presence of C-26 alcohols and C-26aldehydes severely affects the ability of Bgh to germinatein vitro and in vivo, we decided to follow the reduced waxphenotype in a mapping approach and analyze thereafterwhether the gene mutation identified would explain theBgh phenotype.

For mapping, we used the cross between emr1 andGrannenlose Zweizeiligemlo11 that we described previouslyin Jansen et al. (2007). During genetic segregation analysisamong F2 individuals and derived F3 offspring, 15 in-dividuals were found in which higher water retentionand enhanced M. oryzae resistance (emr1 sensu stricto)did not cosegregate, indicating two independent geneloci. For practical reasons, we designated the gene locusresponsible for the wax phenotype as LOWWAX1 (LWA1)and the mutant allele accordingly as lwa1. Plants homo-zygous for one of these two mutations bearing the wild-type allele for the other one were selected from the F3offspring. Inoculation of these plants with Bgh con-idiospores revealed that the lwa1, but not the emr1 allele,is responsible for the impaired Bgh germination (Fig.1B). Segregation of lwa1was followed in 92 F2 plants bywater spraying and differentiation between retention ofsmall or big water droplets on the leaf surface (Fig. 2A).

Figure 1. Investigation of interaction sites of Bgh with different barleygenotypes. Primary leaves of barley plants were inspected at 16 hourspost inoculation (hpi). Progression of prepenetration infection stageswas analyzed for each conidiospore and assigned to different cate-gories as indicated. A, Frequency of different infection stages is givenfor the interaction of Bgh with Ingridmlo5 (mlo5/EMR1/LWA1) ormutant emr1 (mlo5/emr1/lwa1). B, Germination rates of Bgh conidiosporeson barley plants segregating for lwa1 and emr1 alleles. C, Frequency of

different Bgh infection stages on lwa1 mutant plants complemented withthe wild-type LWA1 allele in an IngridMLO or Ingridmlo5 genetic back-ground. Regenerants transformed with a GFP construct served as controls.D, Prepenetration development of Bgh on barley genotypes bearing theLWA1 wild type or the lwa1 mutant allele in the IngridMLO geneticbackground. Bars represent mean values (n = 3)6 SDs, with 100 interactionsites inspected per genotype and per leaf. The experiment was repeatedwith similar results three times (A) or one time (B and D) or with differentleaves of individual events (C). Asterisk indicates significant differences (P,0.7) determined in a Student’s t test.

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The emr1 phenotype was followed by inoculation ofplants with M. oryzae. For single-nucleotide polymor-phism (SNP) genotyping, genomic DNA was extractedfrom all 92 F2 plants and subjected to a 384-multiplexSNP GoldenGate VeraCode barley assay. After genotyp-ing, a linkage map of chromosome 4H containing lwa1,emr1, and linked SNP markers was constructed (Fig. 4;Supplemental Table S1). SNP markers suggested mappositions for the lwa1 and emr1 gene loci on different arms

of the barley chromosome 4H. For additional characteri-zation of the LWA1 gene, we used the colinearity in thegrass genomes (Bennetzen and Freeling, 1997). Using theweb-tool GenomeZipper (Mayer et al., 2009), the rice(Oryza sativa) gene Os03g0219900 (Rice AnnotationProject database O. sativa identifier) was identified asbeing syntenic to the closest barley iSELECT marker4139-888 (syn. BOPA 1_0606). The rice gene next toOs03g0219900 is Os03g0220100 (LOC_Os03g12030), whichencodes a proteinwith a predictedKCS activity. The syntenicregion of barley 4H for this gene was identified as locus1,751 (full length complementary DNA; AK252279.1). This

Figure 2. Leaves of the barley mutant emr1 display an altered epicuticularwax layer. A, Water retention of primary leaves is strongly enhanced onemr1 mutant plants compared with Ingridmlo5. B, SEM of adaxial leafsurfaces of the barley genotypes Ingridmlo5 and emr1. Scale bars = 1 mm.C, Total cuticular waxes were extracted with chloroform from leaves ofIngridmlo5 or emr1 plants and derivatized with bis-(N,N-trimethylsilyl)-trifluoroacetamide. Wax components were quantified by GC-FID, and thetotal amount of waxes was calculated as the sum of single components.Bars represent mean values of three independent measurements (n = 3) 6SDs, with five primary leaves measured per genotype and replicate; asteriskindicates significant difference (P, 0.001) determined in a Student’s t test.[See online article for color version of this figure.]

Figure 3. Quantification of cuticular wax components in different barleygenotypes. Genotypes exhibit the wild-type LWA1 or mutant lwa1 allelein either the IngridMLO or Ingridmlo5 genetic background as indicated.Transgenic plants complemented with the wild-type LWA1 allele in anlwa1 genetic background are marked (compl). Plants transformed withGFP served as controls. Total cuticular wax was extracted from leaves bydipping in chloroform without hydrolyzation. For this analysis, primaryregenerants from independent transformation events selected after DNAgel analysis were used, and wax was extracted from a single leaf of se-lected regenerants. Wax molecule content was determined by GC-MS,and only those showing significant differences between genotypes arepresented.

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sequence corresponds to the barley high-confidence geneMLOC_51583 that was previously referred to asHvCUT1.3(Richardson et al., 2007). Sequencing of a PCR productamplified from genomic DNA from Ingridmlo5 or lwa1plants with primers specific for the LWA1 gene identified asingle-nucleotide acid polymorphism in the mutant geno-type, which led to an amino acid change in the deducedprotein at position L136F (Supplemental Fig. S3). Databaseanalysis revealed a predicted function for the LWA1 geneas KCS, and therefore, the geneHvCUT1.3was renamed asHvKCS6.

Complementation of lwa1

Complementation of the lwa1 mutant was undertakento determine whether the mutant phenotype could be

rescued by constitutive overexpression of the wild-typeHvKCS6 coding sequence from Ingridmlo5 plants andverify the predicted enzymatic function of the HvKCS6protein. Therefore, precultured immature embryos oflwa1 mutant plants were inoculated with either Agro-bacterium spp. strain AGL-1, which harbors a plasmidcontaining the wild-type LWA1 gene under control of theconstitutive maize UBIQUITIN-1 promoter, or the GFPreporter gene under control of the same promoter as acontrol. Selection of putative transformants was doneon hygromycin-containing media; 29 regenerants wereobtained in genotype MLO/lwa1, and 10 regenerantswere obtained in genotype mlo5/lwa1. All LWA1 PCR-positive primary transformants, hemizygous for thetransgene, displayed a restored wild-type epicuticularwax layer as verified bymonitoring of water retention. Incontrast, the transgenic GFP event, which did not harborthe HvKCS6 transgene, behaved like the lwa1 mutant.Based on DNA gel-blot analysis, three independent in-tegration events were selected for both genomic back-grounds (MLO/lwa1 and mlo5/lwa1; Supplemental Fig.S4). GC-MS profiling of wax components revealed thatthe amount of the C-26 alcohol was restored in theseregenerants to the level in IngridMLO or Ingridmlo5plants (Fig. 3). The amount of the C-26 aldehyde was, onaverage, higher for complemented plants compared withwild-type plants, which might be because of the strongconstitutive expression of HvKCS6 and a preferentialconversion of the C-26 alcohol to the corresponding al-dehyde. Interestingly, the amount of C-24 alcohol wasalso elevated in complemented plants compared withlwa1 mutant plants, which might also be an effect of thestrong overexpression of HvKCS6.

In parallel to the GC-MS analysis, a Bgh infectionassay was performed on detached leaves of the pri-mary transformants. A representative result of a singleevent for each group of transformed genotypes (MLO/lwa1 or mlo5/lwa1) is shown in Figure 1C and com-pared with the GFP-transgenic control. Germination ofBgh conidiospores was compromised on the transgenicGFP event to a similar level as on lwa1 mutant plants(Fig. 1A). By contrast, plants transformed with wild-type HvKCS6 in both MLO and mlo5 genetic back-ground showed a significantly lower frequency of notgerminated Bgh conidiospores (10%), which was evenlower than on Ingridmlo5 plants (Fig. 1A). Taken together,our results obtained by complementation verified that thelack of a functional HvKCS6 allele is responsible for thealtered epicuticular wax composition and the compro-mised germination of Bgh conidiospores on lwa1mutantplants.

Introgression of the lwa1 Allele in the MLOGenetic Background

A matter of discussion that could not be answeredaddresses the question of whether the presence of thelwa1 allele only leads to a reduced germination or alsoleads to lower powdery mildew disease severity. Thiswas difficult to examine, because the strong powdery

Figure 4. Linkage map of chromosome 4H based on the F2 populationemr13 Grannenlose Zweizeiligemlo11, including 21 segregating SNPmarkers as well as lwa1 and emr1. The marker positions in centi-morgans and the SNP names are displayed on left and right of the map,respectively. In the genetic linkage map, gene loci of lwa1 and emr1do not cosegregate. The lwa1 locus was mapped close to the iSELECTmarker 4139-888 (syn. Barley Oligo Pool Assay [BOPA] 1_0606).

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mildew resistance allelemlo5 present in lwa1mutant plantsmakes a macroscopic evaluation of mature powdery mil-dew pustules impossible. To assess this important issue,we used a cross between Ingridmlo5/emr1/lwa1 andIngridMLO/EMR1/LWA1 that we described in a previousstudy (Jansen et al., 2007). From this cross, we retrievedplants homozygous for both the wild-type MLO and themutant lwa1 allele. Inoculation of these plants with Bghrevealed a strong reduction in powdery mildew diseasesymptoms (76% less infected leaf area) on lwa1 mutantplants (Fig. 5). Detailed cytological analysis at 48 h postinoculation showed that, on plants of the MLO/LWA1genotype, only 5% of Bgh conidiospores did not germinate,whereas this frequency was 55% for MLO/lwa1 plants(Fig. 1D). This result confirmed the data received with thelwa1 mutant allele in an mlo5 genetic background. Thesignificantly lower germination rate of Bgh conidiosporeson MLO/lwa1 plants resulted in less frequently formedappressoria and haustoria (Supplemental Fig. S5A). How-ever, compared with the percentage of germinated con-idiospores, there was no substantial difference betweenboth genotypes, because one-half of all germinated con-idiospores had established a haustorium at that timepoint. At these successful infection sites, a significantlylower number of haustoria per colony was observed onMLO/lwa1 plants compared with MLO/LWA1 plants at72 hpi (Supplemental Fig. S5B). This ostensible fitnesspenalty did not result in differences in colony size, whichwas evidenced by a similar number of epidermal cellscovered by individual Bgh colonies on both genotypes.Similarly, no differences were found in Bgh colony sizeon both genotypes at later stages of the infection process(96 hpi), and they also did not differ in the number ofconidiophores formed per colony (Supplemental Fig. S5C).From this comprehensive microscopic analysis, it must beconcluded that the reduced number of Bgh pustules onMLO/lwa1 plants is a direct consequence of the severelyreduced germination rate of Bgh conidiospores.

The lwa1 Mutant Allele Also Affects Germination ofNonadapted Powdery Mildew Fungi

Our next goal was to test whether the altered epicutic-ular wax composition of the lwa1 mutant affects prepene-tration infection structures of powdery mildews in general.Thus, we inoculated lwa1mutant plants with conidiosporesof Bgt, which is a nonhost pathogen of barley and thereforeunable to complete its lifecycle. Microscopic analysisrevealed a severe reduction in germination of Bgt con-idiospores on lwa1 leaves comparedwith Ingridmlo5 leaves(Fig. 6A). Thereafter, and similar to the results obtained forBgh, most of the germinated conidiospores developedmature appressoria on both genotypes, indicating that thelwa1 mutation does not affect the efficiency of appresso-rium formation. Next, we investigated whether powderymildew fungi more distantly related to Bgh than Bgt weresimilarly affected in their conidial germination rate on lwa1mutant plants. Accordingly, we inoculated the lwa1mutantwithErysiphe pisi (powderymildewof pea) andGolovinomyces

orontii (powdery mildew of Arabidopsis). Both fungi ex-hibited reduced germination of conidiospores on lwa1mutant plants compared with Ingridmlo5 plants, whichwas evidenced by microscopic analysis (Fig. 6A).

AtCUT1 Was Identified as a Functional Orthologof HvKCS6

Driven by the observation that germination of con-idiospores of adapted as well as nonadapted powderymildew fungi was affected by the altered compositionof epicuticular waxes on the lwa1mutant, we hypothesizedthat wax components, such as hexacosanal, are a generalrequirement for regular germination of powdery mildewconidiospores. BLAST analysis revealed that the geneAt1g68530 (AtCER6/AtCUT1/AtKCS6) is the Arabidopsishomolog toHvKCS6, and phylogenetic analysis supportedthis finding (Supplemental Fig. S6). We used transgenicArabidopsis plants with reduced AtCUT1 expressioncaused by a cosilencing phenomenon after introductionof a 35S:AtCUT1 construct for additional analysis (Millaret al., 1999). In the latter study, it was reported that epi-cuticular waxes are severely reduced on stems of thesetransgenic plants, and primary alcohols with a chain

Figure 5. Development of powdery mildew disease symptoms onbarley plants differing in LWA1/lwa1 alleles. A, Picture of Bgh-infectedprimary leaves of barley cv IngridMLO (MLO/LWA1) or lwa1 mutantplants (MLO/lwa1) at 6 d post inoculation. B, Quantification ofinfected leaf area by image processing. Data are given as means 6 SDs(n = 5). The experiment was repeated two times with similar results.Asterisk indicates significant difference (P = 0.008) determined in aStudent’s t test. [See online article for color version of this figure.]

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length above C-26 and C-28 are less abundant. We inoc-ulated these AtCUT1-silenced plants with nonadaptedpowdery mildew fungi, such as Bgt, Bgh, or E. pisi, whichresulted in each case in a lower germination rate of con-idiospores compared with wild-type Col-0 Arabidopsisplants (Fig. 6B). Again, no effect on the rate of successfullyformed appressoria could be determined. Similarly,inoculation ofAtCUT1-silenced plants with the Arabidopsis-adapted powdery mildew G. orontii revealed a lower co-nidial germination rate and no influence on appressoriumformation (Supplemental Fig. S7A). Moreover, and in ac-cordance with our results received for Bgh on lwa1 barleyplants, a lower germination rate of conidiospores at 16 hpiresulted in reduced disease severity on AtCUT1-silenced

plants, which was evidenced by fewer infection sites withelongated secondary hyphae at 72 hpi (Supplemental Fig.S7B). Altogether, these results suggest that AtCUT1 andHvKCS6 are functional orthologs with respect to their rolein pathogenicity of adapted and nonadapted powderymildew fungi.

DISCUSSION

With the exception of Arabidopsis, for which a genetictoolbox is widely available, identification of genes causinga mutant phenotype is still challenging, especially in ce-reals with rather complex genomes like barley or wheat.The most severe drawback that limits the power of for-ward genetic screens with these important crop plants isthe time-consuming step of generating mapping popula-tions and the subsequent map-based cloning. Certainly,progress made in gene sequencing technologies (next gen-eration sequencing) and bioinformatics, which enable directcomparison of mutant and wild-type genomes, lookspromising and might remarkably shorten the time required(Zuryn et al., 2010). However, although this method wasrecently successfully applied to rice (Nordström et al., 2013),its distribution among a wide range of different plant spe-cies has not been achieved. Here, we report on the iden-tification of a gene causing a reduced wax phenotype inbarley by simultaneous SNPmapping, an integratedmethodat the transition between classical and new gene discov-ery technologies.

Some years ago, we selected the barley emr1 mutant ina screen for genotypes with enhanced resistance againstM. oryzae (Jansen et al., 2007), an upcoming threat causinghead blast in barley and wheat cultivation (Lima andMinella, 2003; Urashima et al., 2004). During mutant char-acterization, we identified a second phenotype, which wasthe enhanced capacity of emr1mutant plants to retain wateron their leaves (Fig. 2A). This observation together with aglossy appearance of leaves reminded us of the pioneeringworks by Lundqvist and von Wettstein (1962) and vonWettstein-Knowles (2001) on eceriferum (cer) barley mutantplants, all of which had defects in wax load of aerial plantorgans. Indeed, SEM and GC-MS analyses of emr1 primaryleaves revealed a decrease in total cuticular waxes (Fig. 2, Band C). On wild-type barley plants, hexacosanol (C-26alcohol), comprising 75% of extractable cuticular wax, isby far the most abundant constituent (Richardson et al.,2005). After performing detailed GC-MS profiling of indi-vidual wax components, we discovered that hexacosanolwas reduced on emr1 mutant leaves by 90% comparedwith the respective control (i.e. Ingridmlo5 plants; Fig. 3;Supplemental Fig. S2) and therefore emr1 exhibits one ofthe most severe waxless phenotypes reported for barleymutants so far.

Because it is known that cuticle components, such as1-hexadecanol or 1,16-hexadecanediol, are crucial (at leaston rice) for appressorium formation of M. oryzae (Gilbertet al., 1996), we inquired whether the reduced waxcoverage is responsible for enhancement of resistanceagainst this fungus. Investigation of a cross between emr1

Figure 6. Prepenetration development of different powdery mildewspecies on barley or Arabidopsis. The interactions of lwa1 barleymutant plants (A) or Arabidopsis 35S:AtCUT1 sense-silenced plants (B)with different species of powdery mildew fungi were microscopicallyinvestigated at 16 hpi. Interaction sites were grouped into differentcategories, from which only those that showed significant differencesare displayed. Bars represent mean values (n = 3) 6 SDs, with 100 inter-action sites inspected per genotype and per leaf. The experiment was re-peated one time with a similar result. Asterisk indicates significantdifferences (P# 0.043) determined in a Student’s t test; Col-0, Columbia-0.

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and a distantly related barley genotype (GrannenloseZweizeiligemlo11) led to the identification of 15 F2 seg-regants in which emr1-dependent resistance againstM. oryzae did not cosegregate with the wax phenotype.This irrefutably showed that both phenotypes are causedby independent mutations. Inoculation of segregants withthe low wax phenotype (lwa1) with the barley powderymildew fungus revealed a significant reduction in thegermination rate of conidiospores, which was indepen-dent of the presence or absence of emr1 or EMR1 alleles(Fig. 1B). This was a unique finding, because a powderymildew phenotype was not reported for any of the barleycer mutants so far. To analyze whether the lwa1 mutationis responsible for the Bgh germination phenotype, an SNP-based mapping approach was performed, which finallyplaced the lwa1 allele on the long arm of chromosome 4H(Fig. 4). Fortunately, a gene in the syntenic region of ricenext to the closest marker was annotated as a wax bio-synthetic pathway gene with a predicted enzymatic func-tion in fatty acid elongation. The respective barley genewas identified as the MLOC_51583 gene of the recentlyreleased barley genome (Mayer et al., 2012). Sequencingconfirmed an SNP between theMLOC_51583 sequences oflwa1 and Ingridmlo5, leading to a Lys to Phe amino acidsubstitution (Supplemental Fig. S3). Also, the barley LWA1protein has a predicted KCS activity. Such KCS activity isrequired for the elongation of VLCFAs, and thus, a loss-of-function mutation would sufficiently explain why the lwa1phenotype is depleted in hexacosanol (Fig. 3). We vali-dated the predicted enzymatic function of LWA1 bycomplementation of lwa1 mutant plants with the LWA1wild-type coding sequence and subsequent GC-MS pro-filing of wax components (Fig. 3). The MLOC_51583 genewas, thereafter, renamed asHvKCS6. Based on the GC-MSdata, which revealed that the depletion in C-26 VLCFAderivatives on lwa1 plants was accompanied by an in-crease in C-24 VLCFA derivatives and their restoration towild-type levels in HvKCS6-complemented transgenics, itbecame evident that HvKCS6 exhibits specificity for theelongation step from C-24 to C-26 wax constituents.Thereby, we functionally proved that HvKCS6 is a con-densing enzyme of the FAE complex and confirmed itsspecificity in chain-length elongation. Besides the com-plementation of the waxless phenotype, germination ofBgh conidiospores also returned to wild-type rates on theregenerants (Fig. 1C), indicating that HvKCS6-derivedVLCFAs have a crucial role in Bgh pathogenicity.A function as KSC in elongation of VLCFAs was also

shown for the Arabidopsis gene At1g68530 (AtCER6/AtCUT1/AtKCS6; Millar et al., 1999). Based on BLASTanalysis, Li et al. (2013) suggested barley EST DQ646644(named in this study HvCER6 and identical to HvCut1.3;Richardson et al., 2007) as a potential homolog forAt1g68530.In their study, Li et al. (2013) placed 27 barley cer loci, noneof which have been cloned so far, on a barley consensusmap. Based on complete linkage,HvCER6 became a primecandidate for ECERIFERUM-ZG (CER-ZG). The localiza-tion of cer-zg on barley chromosome 4H might imply thatcer-zg and lwa1 are identical loci; however, neither thereported phenotype of wax depletion only on upper leaves

for cer-zg mutant plants nor resequencing of HvKCS6 inthe cer-zg mutant background supported this assumption.

An involvement of C-26 VLCFA derivatives (i.e.hexacosanol and hexacosanal) as clues for germinationof Bgh conidiospores had already been suggested byTsuba et al. (2002). Zabka et al. (2008) showed thatglass slides covered with hexacosanal were effective insupporting germination of Bgh conidiospores. Hexa-cosanol, by contrast, was less supportive in this assay.Testing a set of different aldehydes, it became evidentthat n-octacosanal (C-28) followed by n-tetracosanal(C-24) and n-docosanal (C-22) in decreasing order alsostimulated germination of Bgh conidiospores (Hansjakobet al., 2010). It must be noted, however, that from thelatter less efficient compounds, concentrations of one totwo orders of magnitude higher were needed to inducehexacosanal-comparable germination rates. This concen-tration dependency might explain why, on hexacosanal-depleted lwa1 plants, although having a bit higher rate ofn-tetracosanal (Supplemental Fig. S2), a significant re-duction in germination of Bgh conidiospores was found.Hansjakob et al. (2011) performed in vivo experiments onmaize mutants depleted in total leaf cuticular waxes andshowed that chemical complementation of mutant leaveswith hexacosanal elevated the germination rate of Bghconidiospores. Integrating these data from the literaturewith the results obtained in this study, it became evidentthat the depletion in hexacosanal and not the 90% re-duced amount of hexacosanol is responsible for the ger-mination penalty observed for Bgh on lwa1mutant plants.Also, rust fungi from different genera depend on fattyacid-derived signals for differentiation of prepenetrationinfection structures or stomata sensing (Rubiales et al.,2001; Uppalapati et al., 2012). This suggests a commonmechanism by which ancient fungi adapted to plants asforthcoming hosts before ascomycetes split from basidi-omycetes approximately 400 million years ago (Taylorand Berbee, 2006). Inoculation of lwa1mutant plants withP. hordei or P. pachyrhizi did not reveal any influence ofthe altered epicuticular wax composition on the infectionprocess (Supplemental Fig. S8), pointing to specific waxcomponents rather than the general amount of waxes assignals in rust pathogenicity. Alternatively, it could alsobe that those wax components affected by the lwa1 muta-tion do not affect the infection process of the rust patho-gens under investigation.

We broadened our analysis with lwa1 mutant plantswith a set of different powdery mildew species, whichall form a nonhost type of interaction with barley. Ineach case, a significant reduction in germination ofconidiospores was found, irrespective of whether thepathogens came from monocot or dicot host plants(Fig. 6A). Based on quantitative microscopic evaluation,no influence on the formation of other prepenetrationinfection structures was observed. We further extendedthis analysis by performing a complementary series ofexperiments with Arabidopsis plants transcriptionallysilenced for AtCUT1. This was done, because SEMpictures revealed the absence of smooth, platelet-likestructures among epicuticular wax crystals from stems

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of these AtCUT1-silencend plants (Millar et al., 1999),which was reminiscent of the same phenotype on leavesof lwa1 barley mutants, suggesting a conserved functionof HvKCS6 and AtCUT1 in VLCFA biosynthesis.Because no powdery mildew disease phenotype wasreported for the latter Arabidopsis genotype, we starteda series of experiments and inoculated these AtCUT1-silenced plants with monocot and dicot powdery mildewpathogens. Again, this showed the relationship of VLCFA-derivative availability and fully extended germinationof conidiospores (Fig. 6B). Because germination of con-idiospores from Bgt and G. orontii was compromised onArabidopsis AtCUT1 and barley lwa1 mutant plants, itcould be speculated that the missed VLCFA-derivedsignal is identical on both plants. Our results confirmedthat HvKCS6 and AtCUT1 are functional orthologs withrespect to their biosynthetic function and their role inpowdery mildew conidiospore germination. A conservedfunction of genes from barley and Arabidopsis in powderymildew pathogenicity was also observed for barleyrequired for mlo-specified disease resistance2/Arabidopsispenetration deficient1 and HvMLO/AtMLO2, gene pairsinvolved in penetration or postpenetration resistance(Collins et al., 2003; Consonni et al., 2006). The pairHvKCS6/AtCUT1 is another example of such orthologousgenes.

CONCLUSION

Our results show a conserved requirement of plantcuticle-associated signals in pathogenicity of powderymildew pathogens under investigation. This is remark-able, because these pathogen species comprised dicot andmonocot powdery mildews, which build host and non-host interactions with the plant species tested. Given thefact that powdery mildews are separated from each otherbecause of their host specificity, a KCS6-derived germi-nation signal must have been adopted by an ancientpowdery mildew that was pathogenic on the monocot-dicot progenitor. Because it is known that powdery mil-dew spores are short lived, a quick germination on theright surface could have been a crucial step during ad-aptation of a would-be pathogen to its forthcoming host.In this ancient scenario, VLCFA-derived signals are cru-cial clues for host identification that might have been or-chestrated later on by other thigmotactic or chemotacticsignals. It is interesting that powdery mildews keptVLCFA-derived signals, even after speciation and hostrange expansion. During coevolution with their hosts,however, some powdery mildews might have acquiredadditional VLCFA derivatives as signals for acceleratedgermination, a process that would be receivable, becausethe wax composition varies between plant species. Ourresults show the conservation of ancient pathogenicitydeterminants in modern powdery mildew pathogensmore than 140 to 150 million years ago, a time at whichthe monocot-dicot split is expected to have taken place(Chaw et al., 2004). Modifying the epicuticular wax sig-nature might, therefore, be a promising approach towardplant protection.

MATERIALS AND METHODS

Plant Material and Fungal Infection Assays

The barley (Hordeum vulgare) mutant lwa1 was created by chemical mu-tagenesis (NaN3) of the near-isogenic backcross line Ingridmlo5 (obtained fromthe Max Planck Institute for Plant Breeding Research, Cologne, Germany) asdescribed in Jansen et al., 2007. Barley plants segregating for lwa1 and emr1alleles were recruited from the cross emr1 3 Grannenlose Zweizeiligemlo11(Grannenlose Zweizeiligemlo11 was obtained from the Max Planck Institutefor Plant Breeding Research). Plants homozygous for the wild-type MLO andthe mutant lwa1 allele were retrieved from a cross between Ingridmlo5/emr1/lwa1 and IngridMLO/EMR1/LWA1, which was also described in Jansen et al.,2007. Barley plants were grown in a growth chamber (16°C–18°C, 50%–60%relative humidity, and a 16-h photo period; 210 mmol m22 s21). Infection assaywith Magnaporthe oryzae was done as described in Jansen et al., 2007.

Arabidopsis (Arabidopsis thaliana) plants used in this study were transcrip-tionally silenced for AtCUT1 because of sense suppression by the transgene 35S:AtCUT1 (Millar et al., 1999) and the respective wild-type accession Col-0. Plantswere sown in soil, stratified for 2 d at 4°C in darkness, and grown for 2 weeks ina phytotron (22°C, 60% humidity, and an 8.5-h photoperiod; 120 mmol m22 s21).Thereafter, seedlings were transferred to long-day conditions for 4 weeks (22°C,50%–60% humidity, and a 18-h photoperiod; 110 mmol m22 s21).

Powdery mildew (Blumeria graminis f. sp. hordei, Bgh) race K1 (Hinze et al.,1991) was provided by Paul Schulze-Lefert (Max Planck Institute for PlantBreeding Research), whereas B. graminis f. sp. tritici (Bgt) was a field isolatecollected near Aachen, Germany. Both species were propagated in separateplant cabinets (18°C, 65% humidity, and a 16-h photoperiod; 130–150 mmolm22 s21; Bgh on barley cv IngridMLO and Bgt on wheat cv Feldkrone) byweekly inoculation of 7-d-old seedlings. One day before inoculation, oldconidiospores were removed from infected plants by shaking. Adaxial sur-faces of 7-d-old primary barley leaves or 6-week-old Arabidopsis plants wereinoculated by shaking infected plants over horizontally fixed leaves or de-tached leaves on kinetin agar (0.8% [w/v] agar-agar, 2.5 mg mL21 of kinetin) ina settling tower at an average spore density of 1 to 15 conidiospores mm22.After settling of conidiospores for 1 h, plants were returned to growth cabinetsand cultivated under the conditions described above.

Golovinomyces orontii and Erysiphe pisi f. sp. medicaginis were provided byRalph Panstruga (Institute for Biology I, Rheinisch-Westfaelische TechnischeHochschule [RWTH] Aachen University). G. orontii was maintained on Col-0plants (20°C, 55%–60% humidity, and an 8-h photoperiod; 100 mmol m22 s21).Ten to eleven days post inoculation, conidiospores from infected plants wereused to inoculate test plants by brush inoculation. E. pisi was propagated onsusceptible Pisum sativum plants (22°C, 50%–60% humidity, and a 10-h photoperiod;160 mmol m22 s21). Adaxial surfaces of either 7-d-old primary leaves of barley or6-week-old Arabidopsis plants were inoculated by shaking infected plants(10 d post inoculation) over them. Inoculated plant material was incubatedunder conditions as described above.

Puccinia hordei (barley rust) was a field isolate collected near Aachen,Germany that was subcultivated on barley cv IngridMLO in a plant cabinet(18°C, 50%–60% humidity, and a 16-h photoperiod; 140 mmol m22 s21). Forinoculation, 7-d-old barley primary leaves were sprayed with a suspension ofuredospores in freon (1,1,2-trichloro-1,2,2-trefluoroethan) as described in Jansenet al., 2007. Inoculated plants were incubated in a dark moist chamber for 24 hand then kept under the same conditions as described above (Supplemental Fig. S8).

Phakopsora pachyrhizi was isolated from infected plant material collected inBrazil and maintained on the soybean (Glycine max) cv Petrina as described inLoehrer et al., 2008. Uredospore suspension for inoculation was generated bywashing an infected soybean leaf in 0.1% (v/v) Tween 20 in distilled water.Primary leaves of 7-d-old barley plants were inoculated by spraying andplaced in a dark moist chamber for 24 h. Thereafter, plants were kept undernormal growth conditions (Supplemental Fig. S8).

Microscopic and Macroscopic Analyses

For analysis of individual spores by light microscopy (Nikon Eclipse 50i),leaves were cleared with their inoculated surface up on Whatman 3MM papermoistened with ethanol:acetic acid (3:1, v/v) to avoid displacement of non-germinated conidiospores (Lyngkjaer and Carver, 1999). Fungal structureswere stained with ink-acetic acid solution (10% ink in 25% acetic acid, v/v).Images of infection structures (Supplemental Fig. S1) were taken with a LeicaDMR microscope equipped with a JVC digital camera KY-F75U 3-DDC, and soft-ware DISKUS, version 4.81.1027, was used for image acquisition. Quantification of

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infected leaf area by image processing was performed with Adobe Photoshop CS2,version 9.0.

Determination of HvKCS6 Mapping Position

Phenotyping was done with F2 individuals and derived F3 offspring fromthe cross between emr1 and Grannenlose Zweizeiligemlo11 described in Jansenet al., 2007. Therefore, water retention was monitored after spraying of 2-week-old plants with water, and in addition, disease severity was measured afterinoculation with M. oryzae using a detached leaf assay that is also described inJansen et al., 2007. Genomic DNA was isolated from each of these F2 individualsusing 30 to 50 mg of leaf material harvested from 2-week-old seedlings. Thematerial was homogenized using a TissueLyser II (Qiagen), and extraction wascarried out using the BioSprint DNA Plant Kit and the BioSprint 96 workstationfrom the same supplier. Isolated DNA was dissolved in distilled water, and,based on agarose gel electrophoresis, DNA concentration of all samples wasadjusted to approximately 25 ng mL21.

SNP genotyping was carried out at KWS LOCHOW using a 384 iSELECTVeraCode barley chip. After genotyping of 92 F2 plants, a linkage map wasconstructed with JoinMap 4.0 (Kyazma B.V.; Van Ooijen, 2006). Genotype datawere transformed into the mapping data format (AHB [where A representshomozygous emr1; H represents heterozygous; and B represents homozygousGrannenlose Zweizeiligemlo11]). To calculate the genetic linkage map with 92individuals and 161 polymorphic SNPs, the population type and the log ofodds threshold of linkage were set to F2 and $2.0, respectively. Using theseparameters, a linkage map of chromosome 4H containing lwa1, emr1, andlinked SNP markers was constructed.

Construction of the Transformation Vector and Generationof Transgenic Plants

The genomic HvKCS6 sequences of Ingridmlo5 and lwa1 mutant plantswere amplified with forward primer 59-TAGGATCCCAGCACCGTGAACGCA-TAC-39 containing a BamHI restriction site and reverse primer 59-ATGAATTC-CTGGTTTAATTTGGTCGGGGC-39 containing an EcoRI restriction site and clonedinto intermediate vector pUBI-ABM (DNA Cloning Service) between the maize(Zea mays) UBIQUITIN-1 (UBI1) promoter with first intron and the NOPALINESYNTHASE termination sequence ofAgrobacterium tumefaciens. Correct clones wereverified by sequencing, and the whole expression cassette was subcloned intobinary vector p6int-UBI (DNA Cloning Service) through SfiI restriction sites. Thebinary vector was introduced into A. tumefaciens strain AGL-1 by electroporationas described elsewhere (Hensel et al., 2009).

Stable transgenic barley plants were obtained byAgrobacterium spp.-mediatedgene transfer following a method described previously (Hensel et al., 2009) withslight modifications. Briefly, immature embryos of both genotypes were preculturedfor 5 d on blast cell induction mediumwith dicamba at a final concentration of 5 mgL21 at 24°C in the dark (Hensel et al., 2009). After agroinfection, precultured im-mature embryos were cocultured at stacks onmoistened filter paper (300 mL of blastcell conditioned medium; Hensel et al., 2009) in 5.5-cm diameter petri dishes for2 d in the dark. Callus induction was performed on blast cell induction mediumwith either 20 or 50 mg L21 of hygromycin B (Roche Diagnostics). Regeneration tookplace on blast cell recovery medium with 25 mg L21 of hygromycin B at 24°C in thelight (Hensel et al., 2009). In contrast to the previously published method, allregenerants were further processed, excepting that some of the primary transgenicplants may be derived from the same transformation event. Rooting and transferinto soil were the same as described before.

Transgenic events were identified and confirmed by PCR based on primerpairs specific for the junction between ZmUBI-1:hygromycin phosphotransferase(59-TTTAGCCCTGCCTTCATACG-39 and 59-GATTCCTTGCGGTCCGAATG-39)and ZmUBI-1:HvKCS6 (59-TTTAGCCCTGCCTTCATACG-39 and 59-TGCACC-AGCCTGTACTTGG-39) using 100 ng of genomic DNA isolated following theprotocol in Pallotta et al., 2000. To identify siblings, DNA gel-blot analyses wereconducted using an hygromycin phosphotransferase-specific hybridization probethat was constructed with the same primers as for PCR and a DIG-PCR LabelingKit (Roche Diagnostics). Genomic DNA (30 mg) were digested with HindIII andseparated by agarose gel electrophoresis. After transfer on a positively chargednylon membrane (Roche Diagnostics), blots were hybridized and processed fol-lowing the manufacturer’s instructions (Supplemental Fig. S4).

Wax Extraction and GC-MS Analysis

For wax analysis of barley genotypes IngridMLO, Ingridmlo5, and emr1,7-d-old primary leaves were cut off and immediately immersed in chloroform

for 10 s at room temperature. From transgenic plants, either the third or fourthleaf of each individual 5-week-old plant was used for wax analysis. The cor-responding leaf area was determined by scanning of dipped leaves and pixelquantification using Adobe Photoshop CS2, version 9.0. The resulting solutioncontaining the cuticular waxes was spiked with 10 mg of tetracosane (Sigma-Aldrich) as an internal standard. The solvent was evaporated under a streamof nitrogen, and compounds containing free hydroxyl and carboxyl groupswere converted into their trimethylsilyl ethers and esters, respectively, withbis-(N,N-trimethylsilyl)-trifluoroacetamide (Machery-Nagel) in pyridine for 40min at 70°C before GC-MS analysis. Wax constituents were identified by theirelectron impact MS spectra after GC-MS analysis and quantified using anidentical GC system equipped with a flame ionization detector (FID) as de-scribed previously (Richardson et al., 2005).

Phylogenetic Analysis

Protein sequences for barley KCS members were obtained from theEnsemblPlants database (Kinsella et al., 2011), and those for Arabidopsis werefrom The Arabidopsis Information Resource homepage (Lamesch et al., 2012).Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013) byusing the Maximum Likelihood method based on the Jones-Taylor-Thorntonmatrix-based model (Jones et al., 1992) with 1,000 replicates to obtain boot-strap values. Initial trees for the heuristic search were obtained automaticallyby applying Neighbor-Join and BioNJ algorithms to a matrix of pairwisedistances estimated using a Jones-Taylor-Thornton model and then selectingthe topology with superior log-likelihood value. Codon positions includedwere first + second + third + noncoding. All positions containing gaps andmissing data were eliminated. In total, there were 225 positions in the finaldataset. The underlying protein alignment was produced by using the ClustalWmethod implemented in MEGA6 (Supplemental Fig. S6).

Sequence data from this article can be found in the EMBL/GenBank datalibraries or the Arabidopsis Genome Initiative under the following accessionnumbers: barley 3-KETOACYL-CoA SYNTHASE HvCER6/HvCUT1.3/HvKCS6,barley EST DQ646644/full length complementary DNA AK252279.1/MLOC_51583; Arabidopsis 3-KETOACYL-CoA SYNTHASE AtCER6/AtCUT1/AtKCS6, At1g68530; rice gene syntenic to closest barley iSELECT marker 4139-888(syn. BOPA 1_0606), Os03g0219900; and rice 3-KETOACYL-CoA SYNTHASE,Os03g0220100 (LOC_Os03g12030). Accession numbers for barley and ArabidopsisKCS sequences used for phylogenetic analysis are provided in SupplementalFigure S6.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Powdery mildew infection structures on barleyand Arabidopsis.

Supplemental Figure S2. Detailed analysis of cuticular wax profiles fromdifferent barley genotypes.

Supplemental Figure S3. Comparison of HvKCS6 nucleotide sequencesfrom Ingridmlo5 (mlo5) and lwa1 barley genotypes.

Supplemental Figure S4. Southern-blot confirmation of transfer-DNA in-tegration pattern.

Supplemental Figure S5. Microscopic analysis of postpenetration devel-opmental stages of Blumeria graminis f. sp. hordei.

Supplemental Figure S6. Phylogenetic tree of barley and Arabidopsis KCSprotein families.

Supplemental Figure S7. Microscopic analysis of the development of Golo-vinomyces orontii on Arabidopsis transgenic plants (35S:AtCUT1) withsense silencing of AtCUT1.

Supplemental Figure S8. Prepenetration development of a host and nonhostrust species on primary leaves of the lwa1 mutant.

Supplemental Table S1. Genotype data for 21 informative SNPs fromchromosome 4H and emr1 and lwa1 loci across 92 F2 offspring originatingfrom an emr1 3 Grannenlose Zweizeiligemlo11 cross.

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ACKNOWLEDGMENTS

We thank Alan Slusarenko (RWTHAachen University) for helpful discussionsand critical reading of the article, Penny von Wettstein-Knowles (UniversityCopenhagen, Copenhagen, Denmark) for stimulating discussion and suggestions,Jens Leon (University of Bonn) for multiplication of barley material used in thisstudy, Sabine Sommerfeld (Leibniz Institute of Plant Genetics and Crop PlantResearch) for technical assistance in generation of transgenic barley plants, AnjaRheinstädler (RWTH Aachen University) and Ralph Panstruga (RWTH AachenUniversity) for help with the powdery mildew infection assay on Arabidopsis,and Burkhard Schmidt (RWTH Aachen University) for assistance with GC-FIDanalyses of cuticular waxes.

Received July 4, 2014; accepted September 1, 2014; published September 8,2014.

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