Evolutionary Conserved Function of Barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in Providing Wax Signals for Germination of Powdery Mildew Fungi^,,

An orthologous pair of genes from barley and Arabidopsis is involved in long-chain fatty acid signaling that is required for germination of conidia from distantly related powdery mildews.

Abstract

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 identified 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 significantly 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 findings 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 pioneering 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., 2000; Kunst and Samuels, 2003; Bargel et al., 2004). Cutin is a covalently cross-linked polymer made of saturated C-16-hydroxy and partially unsaturated C-18-hydroxy and C-18-epoxy fatty acids (Bargel et al., 2004). Cuticular waxes, by contrast, are complex organic solvent-extractable mixtures of monomeric C-20 to C-60 aliphatics that may include triterpenoids, phenylpropanoids, and flavonoids (Samuels et al., 2008). The composition of intracellular and extracellular waxes is extremely variable and may differ between plant species, organs within a given species, and also, developmental stages of plant organs (Post-Beittenmiller, 1996; Bargel et al., 2004). Wax biosynthesis starts in leucoplasts, the photosynthetically inactive plastids of the epidermis, with de novo synthesis of C-16 and C-18 fatty acids. These are subsequently elongated and modified in the endoplasmic reticulum to, at first, very long-chain fatty 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 extension to VLCFAs are carried out by multienzyme complexes termed fatty acid synthases and fatty acid elongases (FAEs), respectively; both consist of four dissociable enzymes catalyzing consecutive enzymatic reactions (Samuels et al., 2008). For elongation, C-2 units derived from malonyl-CoA are added to fatty acids or VLCFAs by the FAE complex, involving condensation, reduction, dehydration, and a second reduction step. Several cycles are needed to yield chain lengths of, for example, C-24 to C-34. Each of those cycles seems to be carried out by different FAE complexes, which are distinct by individual β-KETOACYL-CoA SYNTHASEs (KCSs), because their specificity is in the KCS condensing enzyme (Samuels et al., 2008; Chen et al., 2011; Haslam and Kunst, 2013). Therefore, it is not surprising that a large family of KCSs exists (e.g. 21 KCS-like sequences have been identified in Arabidopsis [Arabidopsis thaliana]; Kim et al., 2013). However, the function in elongation of epicuticular wax VLCFAs was experimentally verified so far for only two of these enzymes (AtKCS1 and Arabidopsis ECERIFERUM6 [AtCER6]/Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1 [AtCUT1]/AtKCS6; Millar et al., 1999; Todd et al., 1999; Fiebig et al., 2000).

Our current understanding of the wax biosynthetic pathway and the precise function of particular enzymes is still limited, although genetic as well as biochemical approaches have been used widely. Thereby, forward genetic screens profited from the easy to score visual phenotype of mutants with little or no wax coating on aerial plant organs, which appear glossy or glaucous. In barley, these mutants are termed eceriferum (Latin: cera means wax and ferre means to bear), and cer is used as the symbol for the respective gene loci (Lundqvist and von Wettstein, 1962). The Arabidopsis community followed this terminology, and today, 85-cer and 22-cer loci have been identified in barley and Arabidopsis, respectively (Kunst and Samuels, 2003). However, because these mutants are affected in their total wax load rather than differences among wax components, it is unlikely that they cover the whole wax biosynthetic pathway (Post-Beittenmiller, 1998). In Arabidopsis, this gap was closed by reverse genetic approaches, which enabled the identification of additional genes involved in cuticular wax biosynthesis (Kunst and Samuels, 2003). Sequence homologies to some of these genes were used to clone candidate genes of barley (Richardson et al., 2007). However, because forward and reverse genetic data are missing, gene-function relationships in barley are still speculative.

Airborne plant pathogens, regardless of whether they are dispersed by water or wind, have to cope with the problem of adhesion to the waxy surface of their host plants. In the case of water-disseminated pathogens, such as Magnaporthe oryzae or Colletotrichum graminicola, this is achieved by release of glue, whereas for wind-disseminated pathogens, like powdery mildews, the initial contact can be secured by hydrophobic interactions between the conidiospore and the leaf surface (Epstein and Nicholson, 2006). Nevertheless, conidial extracellular material might also help in this case to attach a conidiospore to the plant surface (Wright et al., 2002). After attachment, pathogens have to overcome the cuticle and the epidermal cell wall barriers to reach the nutrient pool of the plant interior. This process is referred to as penetration, and it might involve the generation of high turgor pressure in dedicated cells (so-called appressoria) to mechanically breach the cell wall, which is eventually supported by the activity of hydrolytic enzymes (Tucker and Talbot, 2001). For example, in Blumeria graminis f. sp. hordei (Bgh; the barley powdery mildew fungus), a combination of turgor-driven and enzymatic-supported penetration has been suggested (Pryce-Jones et al., 1999). Generally, powdery mildews are highly specialized biotrophic pathogens infecting and reproducing only on living tissue of a limited range of plant species (Both and Spanu, 2004). The disease cycle of Bgh, which uses barley as the sole host, starts with the attachment of a conidiospore to the leaf surface and the formation of a primary germ tube (Carver et al., 1995). In addition, an appressorial germ tube is formed, giving rise at its tip to an appressorium from which the fungus penetrates the underlying tissue. In the case of success, the fungus invades an epidermal cell and forms his feeding organ, the so-called haustorium, while leaving the plasma membrane intact. Thereafter, the pathogen develops secondary hyphae on the leaf surface and continues to penetrate neighboring epidermal cells. The fungus completes its lifecycle by building conidial mother cells, and novel conidiospores emerge (Eichmann and Hückelhoven, 2008). Right after initial contact, Bgh conidiospores prepare an infection court at the interface to their host, and Epstein and Nicholson (2006) speculated that, at this space of tight adherence, the concentration of lytic enzymes, such as cutinases (Pascholati et al., 1992), effectively could be maintained at higher levels. This enzymatic activity may lead to the release of monomeric or oligomeric degradation products (e.g. cutin monomers), which can act as damage-associated molecular patterns and trigger defense responses (Schweizer et al., 1996; Tucker and Talbot, 2001). The involvement of epicuticular wax components in defense was shown in a recent study, in which silencing of a cytochrome P450 gene, involved in the generation of VLCFA derivatives such as secondary alcohols and ketones, diminished penetration resistance of barley against M. oryzae (Delventhal et al., 2014). By contrast, cutin monomers may also contribute to disease susceptibility, because, for example, in Bgh and Ustilago maydis, they play a role in appressorial germ tube and appressorium formation, respectively (Francis et al., 1996; Mendoza-Mendoza et al., 2009). For the wheat powdery mildew fungus B. graminis f. sp. tritici (Bgt), it was shown that the expression of a secreted lipase involved in the release of epicuticular wax components from infected wheat (Triticum aestivum) leaves was important for fungal adhesion and development (Feng et al., 2009). Additional fatty acid components supporting pathogen development rather than acting as damage-associated molecular patterns are long-chain and very long-chain alcohols and aldehydes from the epicuticular wax layer, which play a crucial role in spore germination of Bgh (Hansjakob et al., 2010), Puccinia emaculata, Phakopsora pachyrhizi, and Colletotrichum trifolii (Uppalapati et al., 2012). For the latter three pathogens, a gene involved in the generation of respective wax compounds was cloned from Medicago truncatula and turned out to be a transcription factor affecting wax biosynthetic genes. For Bgh, however, no wax biosynthetic pathway gene involved in spore germination has been cloned so far from its intrinsic barley host. Thus, evidence for the involvement of VLCFA derivatives in Bgh germination comes from experiments with nonhost wax mutant plants, such as maize (Zea mays) or Arabidopsis, and chemical complementation with particular VLCFA derivatives (Hansjakob et al., 2010; Weis et al., 2014). An important issue that, therefore, could not be touched so far is the question of whether the reduced Bgh conidial germination rate affects disease severity.

Here, we close this long-lasting gap by identifying the barley KCS gene HvKCS6 as being required for germination of Bgh conidiospores on its host. We verified (by genomic complementation of the respective mutant and gas chromatography [GC] -mass spectrometry [MS] analysis of wax components) that this gene encodes a condensing enzyme that is part of the fatty acid elongation complex and has a presumed specificity for elongation of C-24 to C-26 VLCFAs. Comparative analyses with Arabidopsis revealed a conserved function of the orthologous gene in providing essential signals for germination of conidiospores from different powdery mildew species. Using compatible host-pathogen combinations, we showed that, on barley and Arabidopsis wax mutant plants, a reduced germination rate of powdery mildew conidiospores finally resulted in less frequently formed disease symptoms, thus opening the road, to our knowledge, to a new breeding trait.

RESULTS

Germination of Bgh Conidiospores Is Compromised on Barley Mutant Enhanced Magnaporthe Resistance Gene1

The barley mutant enhanced Magnaporthe resistance gene1 (emr1), generated in our laboratory, was identified in a suppressor screen for restoration of resistance against M. oryzae in the hypersusceptible genetic background mildew resistance locus O allele5 (Ingridmlo5; Jansen et al., 2007). Based on visual scoring of disease symptoms, no phenotypic response was observed against other important barley leaf pathogens (e.g. powdery mildew, net blotch [Drechslera teres], leaf scald [Rhynchosporium secalis], or rust [Puccinia hordei]; Jansen and Schaffrath, 2009). For powdery mildew, however, the presence of the strong resistance allele mlo5 could have masked potential effects. Therefore, we reevaluated the interaction between Bgh and emr1 mutant plants using a microscopic assay and analyzed the formation of initial infection structures as depicted in Supplemental Figure S1. In this experiment, the percentage of conidiospores that did not germinate on leaves of emr1 mutant plants was almost 2 times as high (33%) compared with those on leaves of its ancestor Ingridmlo5 (18%; Fig. 1A). Germinated conidiospores gave rise to mature appressoria at a similar rate on both genotypes (i.e. on Ingridmlo5 plants, 82% germination and 72% appressoria; on emr1 mutant plants, 67% germination and 55% appressoria). Thus, apart from compromised germination, no additional differences were found in the prepenetration process of Bgh. These results show that the emr1 mutant exhibits two different phenotypes, one of which is the enhanced resistance against M. oryzae and the other is a reduction in the germination frequency of Bgh conidiospores. The following experiments were designed to answer the question of whether both phenotypes are conferred by the same mutation and identify the underlying gene(s).

Figure 1.

Investigation of interaction sites of Bgh with different barley genotypes. Primary leaves of barley plants were inspected at 16 hours post inoculation (hpi). Progression of prepenetration infection stages was analyzed for each conidiospore and assigned to different categories as indicated. A, Frequency of different infection stages is given for the interaction of Bgh with Ingridmlo5 (mlo5/EMR1/LWA1) or mutant emr1 (mlo5/emr1/lwa1). B, Germination rates of Bgh conidiospores on barley plants segregating for lwa1 and emr1 alleles. C, Frequency of different Bgh infection stages on lwa1 mutant plants complemented with the wild-type LWA1 allele in an IngridMLO or Ingridmlo5 genetic background. Regenerants transformed with a GFP construct served as controls. D, Prepenetration development of Bgh on barley genotypes bearing the LWA1 wild type or the lwa1 mutant allele in the IngridMLO genetic background. Bars represent mean values (n = 3) ± sds, with 100 interaction sites inspected per genotype and per leaf. The experiment was repeated with similar results three times (A) or one time (B and D) or with different leaves of individual events (C). Asterisk indicates significant differences (P < 0.7) determined in a Student’s t test.

emr1 Is Depleted in Leaf Surface Waxes

During inoculation, a higher capacity for water retention was observed on leaves of the emr1 mutant compared with other barley cultivars (Fig. 2A). This observation, together with a glossy appearance of emr1 leaves, was reminiscent of barley eceriferum (cer) mutant plants with altered leaf wax coating (Post-Beittenmiller, 1996). We applied scanning electron microscopy (SEM) to further investigate this phenomenon and found a strong reduction of wax crystals on the leaf surfaces of emr1 mutants compared with those of Ingridmlo5 plants (Fig. 2B). Taking a closer look, it appears as if particular smooth, platelet-like structures of wax crystals are absent on mutant leaves. Applying GC-MS, the difference between Ingridmlo5 and emr1 mutant plants in total content of cuticular leaf wax could be quantified to 10 and 2.4 µg cm⁻², respectively (Fig. 2C).

Figure 2.

Leaves of the barley mutant emr1 display an altered epicuticular wax layer. A, Water retention of primary leaves is strongly enhanced on emr1 mutant plants compared with Ingridmlo5. B, SEM of adaxial leaf surfaces of the barley genotypes Ingridmlo5 and emr1. Scale bars = 1 µm. C, Total cuticular waxes were extracted with chloroform from leaves of Ingridmlo5 or emr1 plants and derivatized with bis-(N,N-trimethylsilyl)-trifluoroacetamide. Wax components were quantified by GC-FID, and the total amount of waxes was calculated as the sum of single components. Bars represent mean values of three independent measurements (n = 3) ± sds, with five primary leaves measured per genotype and replicate; asterisk indicates significant difference (P < 0.001) determined in a Student's t test. [See online article for color version of this figure.]

Based on the SEM pictures, we speculated that emr1 mutants lack particular components of the epicuticular leaf wax. We followed this thought in more depth by detailed GC-MS analysis of individual wax components. Strikingly, we determined that the most abundant component among barley cuticular leaf waxes, the C-26 alcohol (hexacosanol), was reduced by 90% on primary leaves of emr1 mutant plants compared with IngridMLO and Ingridmlo5 plants, respectively (Fig. 3; Supplemental Fig. S2). Similar to the decrease in hexacosanol content, the amount of the C-26 aldehyde (hexacosanal) dropped from 0.3 µg cm⁻² on Ingridmlo5 plants to 0.007 µg cm⁻² on emr1 plants. Concomitant with the reduction in wax components with C-26 chain lengths, an increase of the C-24 alcohol (tetracosanol) was measured on emr1 (Fig. 3), suggesting a block in elongation of VLCFAs from C-24 to C-26 chain lengths in emr1 plants. This hypothesis is supported by the observation that C-26-based components were also absent in wax esters of emr1 mutant plants (Supplemental Fig. S2). Interestingly, emr1 plants seem to compensate for the lack of C26 constituents in esters by forming novel alcohol-acid combinations to keep the total chain length (as the sum of alcohol and acid). For example, in Ingridmlo5 plants, the ester with a chain length of C-46 is built from the C-26 alcohol and the C-20 acid, whereas in emr1 mutant plants, the C-46 ester is made from the C-24 alcohol and the C-22 acid or the C-22 alcohol and the C-24 acid (Supplemental Fig. S2).

Figure 3.

Quantification of cuticular wax components in different barley genotypes. Genotypes exhibit the wild-type LWA1 or mutant lwa1 allele in either the IngridMLO or Ingridmlo5 genetic background as indicated. Transgenic plants complemented with the wild-type LWA1 allele in an lwa1 genetic background are marked (compl). Plants transformed with GFP served as controls. Total cuticular wax was extracted from leaves by dipping in chloroform without hydrolyzation. For this analysis, primary regenerants from independent transformation events selected after DNA gel analysis were used, and wax was extracted from a single leaf of selected regenerants. Wax molecule content was determined by GC-MS, and only those showing significant differences between genotypes are presented.

Mapping and In Silico Identification of the Gene Responsible for the Wax Phenotype

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

For mapping, we used the cross between emr1 and Grannenlose Zweizeiligemlo11 that we described previously in Jansen et al. (2007). During genetic segregation analysis among F2 individuals and derived F3 offspring, 15 individuals were found in which higher water retention and enhanced M. oryzae resistance (emr1 sensu stricto) did not cosegregate, indicating two independent gene loci. For practical reasons, we designated the gene locus responsible for the wax phenotype as LOW WAX1 (LWA1) and the mutant allele accordingly as lwa1. Plants homozygous for one of these two mutations bearing the wild-type allele for the other one were selected from the F3 offspring. Inoculation of these plants with Bgh conidiospores revealed that the lwa1, but not the emr1 allele, is responsible for the impaired Bgh germination (Fig. 1B). Segregation of lwa1 was followed in 92 F2 plants by water spraying and differentiation between retention of small or big water droplets on the leaf surface (Fig. 2A). The emr1 phenotype was followed by inoculation of plants with M. oryzae. For single-nucleotide polymorphism (SNP) genotyping, genomic DNA was extracted from all 92 F2 plants and subjected to a 384-multiplex SNP GoldenGate VeraCode barley assay. After genotyping, a linkage map of chromosome 4H containing lwa1, emr1, and linked SNP markers was constructed (Fig. 4; Supplemental Table S1). SNP markers suggested map positions for the lwa1 and emr1 gene loci on different arms of the barley chromosome 4H. For additional characterization of the LWA1 gene, we used the colinearity in the grass genomes (Bennetzen and Freeling, 1997). Using the web-tool GenomeZipper (Mayer et al., 2009), the rice (Oryza sativa) gene Os03g0219900 (Rice Annotation Project database O. sativa identifier) was identified as being syntenic to the closest barley iSELECT marker 4139-888 (syn. BOPA 1_0606). The rice gene next to Os03g0219900 is Os03g0220100 (LOC_Os03g12030), which encodes a protein with a predicted KCS activity. The syntenic region of barley 4H for this gene was identified as locus 1,751 (full length complementary DNA; AK252279.1). This sequence corresponds to the barley high-confidence gene MLOC_51583 that was previously referred to as HvCUT1.3 (Richardson et al., 2007). Sequencing of a PCR product amplified from genomic DNA from Ingridmlo5 or lwa1 plants with primers specific for the LWA1 gene identified a single-nucleotide acid polymorphism in the mutant genotype, which led to an amino acid change in the deduced protein at position ^L136^F (Supplemental Fig. S3). Database analysis revealed a predicted function for the LWA1 gene as KCS, and therefore, the gene HvCUT1.3 was renamed as HvKCS6.

Figure 4.

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

Complementation of lwa1

Complementation of the lwa1 mutant was undertaken to determine whether the mutant phenotype could be rescued by constitutive overexpression of the wild-type HvKCS6 coding sequence from Ingridmlo5 plants and verify the predicted enzymatic function of the HvKCS6 protein. Therefore, precultured immature embryos of lwa1 mutant plants were inoculated with either Agrobacterium spp. strain AGL-1, which harbors a plasmid containing the wild-type LWA1 gene under control of the constitutive maize UBIQUITIN-1 promoter, or the GFP reporter gene under control of the same promoter as a control. Selection of putative transformants was done on hygromycin-containing media; 29 regenerants were obtained in genotype MLO/lwa1, and 10 regenerants were obtained in genotype mlo5/lwa1. All LWA1 PCR-positive primary transformants, hemizygous for the transgene, displayed a restored wild-type epicuticular wax layer as verified by monitoring of water retention. In contrast, the transgenic GFP event, which did not harbor the HvKCS6 transgene, behaved like the lwa1 mutant. Based on DNA gel-blot analysis, three independent integration events were selected for both genomic backgrounds (MLO/lwa1 and mlo5/lwa1; Supplemental Fig. S4). GC-MS profiling of wax components revealed that the amount of the C-26 alcohol was restored in these regenerants to the level in IngridMLO or Ingridmlo5 plants (Fig. 3). The amount of the C-26 aldehyde was, on average, higher for complemented plants compared with wild-type plants, which might be because of the strong constitutive expression of HvKCS6 and a preferential conversion of the C-26 alcohol to the corresponding aldehyde. Interestingly, the amount of C-24 alcohol was also elevated in complemented plants compared with lwa1 mutant plants, which might also be an effect of the strong overexpression of HvKCS6.

In parallel to the GC-MS analysis, a Bgh infection assay was performed on detached leaves of the primary transformants. A representative result of a single event for each group of transformed genotypes (MLO/lwa1 or mlo5/lwa1) is shown in Figure 1C and compared with the GFP-transgenic control. Germination of Bgh conidiospores was compromised on the transgenic GFP 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 background showed a significantly lower frequency of not germinated Bgh conidiospores (10%), which was even lower than on Ingridmlo5 plants (Fig. 1A). Taken together, our results obtained by complementation verified that the lack of a functional HvKCS6 allele is responsible for the altered epicuticular wax composition and the compromised germination of Bgh conidiospores on lwa1 mutant plants.

Introgression of the lwa1 Allele in the MLO Genetic Background

A matter of discussion that could not be answered addresses the question of whether the presence of the lwa1 allele only leads to a reduced germination or also leads to lower powdery mildew disease severity. This was difficult to examine, because the strong powdery mildew resistance allele mlo5 present in lwa1 mutant plants makes a macroscopic evaluation of mature powdery mildew pustules impossible. To assess this important issue, we used a cross between Ingridmlo5/emr1/lwa1 and IngridMLO/EMR1/LWA1 that we described in a previous study (Jansen et al., 2007). From this cross, we retrieved plants homozygous for both the wild-type MLO and the mutant lwa1 allele. Inoculation of these plants with Bgh revealed a strong reduction in powdery mildew disease symptoms (76% less infected leaf area) on lwa1 mutant plants (Fig. 5). Detailed cytological analysis at 48 h post inoculation showed that, on plants of the MLO/LWA1 genotype, 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 the lwa1 mutant allele in an mlo5 genetic background. The significantly lower germination rate of Bgh conidiospores on MLO/lwa1 plants resulted in less frequently formed appressoria and haustoria (Supplemental Fig. S5A). However, compared with the percentage of germinated conidiospores, there was no substantial difference between both genotypes, because one-half of all germinated conidiospores had established a haustorium at that time point. At these successful infection sites, a significantly lower number of haustoria per colony was observed on MLO/lwa1 plants compared with MLO/LWA1 plants at 72 hpi (Supplemental Fig. S5B). This ostensible fitness penalty did not result in differences in colony size, which was evidenced by a similar number of epidermal cells covered by individual Bgh colonies on both genotypes. Similarly, no differences were found in Bgh colony size on both genotypes at later stages of the infection process (96 hpi), and they also did not differ in the number of conidiophores formed per colony (Supplemental Fig. S5C). From this comprehensive microscopic analysis, it must be concluded that the reduced number of Bgh pustules on MLO/lwa1 plants is a direct consequence of the severely reduced germination rate of Bgh conidiospores.

Figure 5.

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

The lwa1 Mutant Allele Also Affects Germination of Nonadapted Powdery Mildew Fungi

Our next goal was to test whether the altered epicuticular wax composition of the lwa1 mutant affects prepenetration infection structures of powdery mildews in general. Thus, we inoculated lwa1 mutant plants with conidiospores of Bgt, which is a nonhost pathogen of barley and therefore unable to complete its lifecycle. Microscopic analysis revealed a severe reduction in germination of Bgt conidiospores on lwa1 leaves compared with Ingridmlo5 leaves (Fig. 6A). Thereafter, and similar to the results obtained for Bgh, most of the germinated conidiospores developed mature appressoria on both genotypes, indicating that the lwa1 mutation does not affect the efficiency of appressorium formation. Next, we investigated whether powdery mildew fungi more distantly related to Bgh than Bgt were similarly affected in their conidial germination rate on lwa1 mutant plants. Accordingly, we inoculated the lwa1 mutant with Erysiphe pisi (powdery mildew of pea) and Golovinomyces orontii (powdery mildew of Arabidopsis). Both fungi exhibited reduced germination of conidiospores on lwa1 mutant plants compared with Ingridmlo5 plants, which was evidenced by microscopic analysis (Fig. 6A).

Figure 6.

Prepenetration development of different powdery mildew species on barley or Arabidopsis. The interactions of lwa1 barley mutant plants (A) or Arabidopsis 35S:AtCUT1 sense-silenced plants (B) with different species of powdery mildew fungi were microscopically investigated at 16 hpi. Interaction sites were grouped into different categories, from which only those that showed significant differences are displayed. Bars represent mean values (n = 3) ± sds, with 100 interaction sites inspected per genotype and per leaf. The experiment was repeated one time with a similar result. Asterisk indicates significant differences (P ≤ 0.043) determined in a Student’s t test; Col-0, Columbia-0.

AtCUT1 Was Identified as a Functional Ortholog of HvKCS6

Driven by the observation that germination of conidiospores of adapted as well as nonadapted powdery mildew fungi was affected by the altered composition of epicuticular waxes on the lwa1 mutant, we hypothesized that wax components, such as hexacosanal, are a general requirement for regular germination of powdery mildew conidiospores. BLAST analysis revealed that the gene At1g68530 (AtCER6/AtCUT1/AtKCS6) is the Arabidopsis homolog to HvKCS6, and phylogenetic analysis supported this finding (Supplemental Fig. S6). We used transgenic Arabidopsis plants with reduced AtCUT1 expression caused by a cosilencing phenomenon after introduction of a 35S:AtCUT1 construct for additional analysis (Millar et al., 1999). In the latter study, it was reported that epicuticular waxes are severely reduced on stems of these transgenic plants, and primary alcohols with a chain length above C-26 and C-28 are less abundant. We inoculated these AtCUT1-silenced plants with nonadapted powdery mildew fungi, such as Bgt, Bgh, or E. pisi, which resulted in each case in a lower germination rate of conidiospores compared with wild-type Col-0 Arabidopsis plants (Fig. 6B). Again, no effect on the rate of successfully formed appressoria could be determined. Similarly, inoculation of AtCUT1-silenced plants with the Arabidopsis-adapted powdery mildew G. orontii revealed a lower conidial germination rate and no influence on appressorium formation (Supplemental Fig. S7A). Moreover, and in accordance with our results received for Bgh on lwa1 barley plants, a lower germination rate of conidiospores at 16 hpi resulted in reduced disease severity on AtCUT1-silenced plants, which was evidenced by fewer infection sites with elongated secondary hyphae at 72 hpi (Supplemental Fig. S7B). Altogether, these results suggest that AtCUT1 and HvKCS6 are functional orthologs with respect to their role in pathogenicity of adapted and nonadapted powdery mildew fungi.

DISCUSSION

With the exception of Arabidopsis, for which a genetic toolbox is widely available, identification of genes causing a mutant phenotype is still challenging, especially in cereals with rather complex genomes like barley or wheat. The most severe drawback that limits the power of forward genetic screens with these important crop plants is the time-consuming step of generating mapping populations and the subsequent map-based cloning. Certainly, progress made in gene sequencing technologies (next generation sequencing) and bioinformatics, which enable direct comparison of mutant and wild-type genomes, looks promising and might remarkably shorten the time required (Zuryn et al., 2010). However, although this method was recently successfully applied to rice (Nordström et al., 2013), its distribution among a wide range of different plant species has not been achieved. Here, we report on the identification of a gene causing a reduced wax phenotype in barley by simultaneous SNP mapping, an integrated method at the transition between classical and new gene discovery technologies.

Some years ago, we selected the barley emr1 mutant in a screen for genotypes with enhanced resistance against M. oryzae (Jansen et al., 2007), an upcoming threat causing head blast in barley and wheat cultivation (Lima and Minella, 2003; Urashima et al., 2004). During mutant characterization, we identified a second phenotype, which was the enhanced capacity of emr1 mutant plants to retain water on their leaves (Fig. 2A). This observation together with a glossy appearance of leaves reminded us of the pioneering works by Lundqvist and von Wettstein (1962) and von Wettstein-Knowles (2001) on eceriferum (cer) barley mutant plants, all of which had defects in wax load of aerial plant organs. Indeed, SEM and GC-MS analyses of emr1 primary leaves revealed a decrease in total cuticular waxes (Fig. 2, B and C). On wild-type barley plants, hexacosanol (C-26 alcohol), comprising 75% of extractable cuticular wax, is by far the most abundant constituent (Richardson et al., 2005). After performing detailed GC-MS profiling of individual wax components, we discovered that hexacosanol was reduced on emr1 mutant leaves by 90% compared with the respective control (i.e. Ingridmlo5 plants; Fig. 3; Supplemental Fig. S2) and therefore emr1 exhibits one of the most severe waxless phenotypes reported for barley mutants so far.

Because it is known that cuticle components, such as 1-hexadecanol or 1,16-hexadecanediol, are crucial (at least on rice) for appressorium formation of M. oryzae (Gilbert et al., 1996), we inquired whether the reduced wax coverage is responsible for enhancement of resistance against this fungus. Investigation of a cross between emr1 and a distantly related barley genotype (Grannenlose Zweizeiligemlo11) led to the identification of 15 F2 segregants in which emr1-dependent resistance against M. oryzae did not cosegregate with the wax phenotype. This irrefutably showed that both phenotypes are caused by independent mutations. Inoculation of segregants with the low wax phenotype (lwa1) with the barley powdery mildew fungus revealed a significant reduction in the germination rate of conidiospores, which was independent of the presence or absence of emr1 or EMR1 alleles (Fig. 1B). This was a unique finding, because a powdery mildew phenotype was not reported for any of the barley cer mutants so far. To analyze whether the lwa1 mutation is responsible for the Bgh germination phenotype, an SNP-based mapping approach was performed, which finally placed the lwa1 allele on the long arm of chromosome 4H (Fig. 4). Fortunately, a gene in the syntenic region of rice next to the closest marker was annotated as a wax biosynthetic pathway gene with a predicted enzymatic function in fatty acid elongation. The respective barley gene was identified as the MLOC_51583 gene of the recently released barley genome (Mayer et al., 2012). Sequencing confirmed an SNP between the MLOC_51583 sequences of lwa1 and Ingridmlo5, leading to a Lys to Phe amino acid substitution (Supplemental Fig. S3). Also, the barley LWA1 protein has a predicted KCS activity. Such KCS activity is required for the elongation of VLCFAs, and thus, a loss-of-function mutation would sufficiently explain why the lwa1 phenotype is depleted in hexacosanol (Fig. 3). We validated the predicted enzymatic function of LWA1 by complementation of lwa1 mutant plants with the LWA1 wild-type coding sequence and subsequent GC-MS profiling of wax components (Fig. 3). The MLOC_51583 gene was, thereafter, renamed as HvKCS6. Based on the GC-MS data, which revealed that the depletion in C-26 VLCFA derivatives on lwa1 plants was accompanied by an increase in C-24 VLCFA derivatives and their restoration to wild-type levels in HvKCS6-complemented transgenics, it became evident that HvKCS6 exhibits specificity for the elongation step from C-24 to C-26 wax constituents. Thereby, we functionally proved that HvKCS6 is a condensing enzyme of the FAE complex and confirmed its specificity in chain-length elongation. Besides the complementation of the waxless phenotype, germination of Bgh conidiospores also returned to wild-type rates on the regenerants (Fig. 1C), indicating that HvKCS6-derived VLCFAs 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 BLAST analysis, 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 for At1g68530. In their study, Li et al. (2013) placed 27 barley cer loci, none of which have been cloned so far, on a barley consensus map. Based on complete linkage, HvCER6 became a prime candidate for ECERIFERUM-ZG (CER-ZG). The localization of cer-zg on barley chromosome 4H might imply that cer-zg and lwa1 are identical loci; however, neither the reported phenotype of wax depletion only on upper leaves for cer-zg mutant plants nor resequencing of HvKCS6 in the cer-zg mutant background supported this assumption.

An involvement of C-26 VLCFA derivatives (i.e. hexacosanol and hexacosanal) as clues for germination of Bgh conidiospores had already been suggested by Tsuba et al. (2002). Zabka et al. (2008) showed that glass slides covered with hexacosanal were effective in supporting germination of Bgh conidiospores. Hexacosanol, by contrast, was less supportive in this assay. Testing a set of different aldehydes, it became evident that n-octacosanal (C-28) followed by n-tetracosanal (C-24) and n-docosanal (C-22) in decreasing order also stimulated germination of Bgh conidiospores (Hansjakob et al., 2010). It must be noted, however, that from the latter less efficient compounds, concentrations of one to two orders of magnitude higher were needed to induce hexacosanal-comparable germination rates. This concentration dependency might explain why, on hexacosanal-depleted lwa1 plants, although having a bit higher rate of n-tetracosanal (Supplemental Fig. S2), a significant reduction in germination of Bgh conidiospores was found. Hansjakob et al. (2011) performed in vivo experiments on maize mutants depleted in total leaf cuticular waxes and showed that chemical complementation of mutant leaves with hexacosanal elevated the germination rate of Bgh conidiospores. Integrating these data from the literature with the results obtained in this study, it became evident that the depletion in hexacosanal and not the 90% reduced amount of hexacosanol is responsible for the germination penalty observed for Bgh on lwa1 mutant plants. Also, rust fungi from different genera depend on fatty acid-derived signals for differentiation of prepenetration infection structures or stomata sensing (Rubiales et al., 2001; Uppalapati et al., 2012). This suggests a common mechanism by which ancient fungi adapted to plants as forthcoming hosts before ascomycetes split from basidiomycetes approximately 400 million years ago (Taylor and Berbee, 2006). Inoculation of lwa1 mutant plants with P. hordei or P. pachyrhizi did not reveal any influence of the altered epicuticular wax composition on the infection process (Supplemental Fig. S8), pointing to specific wax components rather than the general amount of waxes as signals in rust pathogenicity. Alternatively, it could also be that those wax components affected by the lwa1 mutation do not affect the infection process of the rust pathogens under investigation.

We broadened our analysis with lwa1 mutant plants with a set of different powdery mildew species, which all form a nonhost type of interaction with barley. In each case, a significant reduction in germination of conidiospores was found, irrespective of whether the pathogens came from monocot or dicot host plants (Fig. 6A). Based on quantitative microscopic evaluation, no influence on the formation of other prepenetration infection structures was observed. We further extended this analysis by performing a complementary series of experiments with Arabidopsis plants transcriptionally silenced for AtCUT1. This was done, because SEM pictures revealed the absence of smooth, platelet-like structures among epicuticular wax crystals from stems of these AtCUT1-silencend plants (Millar et al., 1999), which was reminiscent of the same phenotype on leaves of lwa1 barley mutants, suggesting a conserved function of HvKCS6 and AtCUT1 in VLCFA biosynthesis. Because no powdery mildew disease phenotype was reported for the latter Arabidopsis genotype, we started a series of experiments and inoculated these AtCUT1-silenced plants with monocot and dicot powdery mildew pathogens. Again, this showed the relationship of VLCFA-derivative availability and fully extended germination of conidiospores (Fig. 6B). Because germination of conidiospores from Bgt and G. orontii was compromised on Arabidopsis AtCUT1 and barley lwa1 mutant plants, it could be speculated that the missed VLCFA-derived signal is identical on both plants. Our results confirmed that HvKCS6 and AtCUT1 are functional orthologs with respect to their biosynthetic function and their role in powdery mildew conidiospore germination. A conserved function of genes from barley and Arabidopsis in powdery mildew pathogenicity was also observed for barley required for mlo-specified disease resistance2/Arabidopsis penetration deficient1 and HvMLO/AtMLO2, gene pairs involved in penetration or postpenetration resistance (Collins et al., 2003; Consonni et al., 2006). The pair HvKCS6/AtCUT1 is another example of such orthologous genes.

CONCLUSION

Our results show a conserved requirement of plant cuticle-associated signals in pathogenicity of powdery mildew pathogens under investigation. This is remarkable, because these pathogen species comprised dicot and monocot powdery mildews, which build host and nonhost interactions with the plant species tested. Given the fact that powdery mildews are separated from each other because of their host specificity, a KCS6-derived germination signal must have been adopted by an ancient powdery mildew that was pathogenic on the monocot-dicot progenitor. Because it is known that powdery mildew spores are short lived, a quick germination on the right surface could have been a crucial step during adaptation of a would-be pathogen to its forthcoming host. In this ancient scenario, VLCFA-derived signals are crucial clues for host identification that might have been orchestrated later on by other thigmotactic or chemotactic signals. It is interesting that powdery mildews kept VLCFA-derived signals, even after speciation and host range expansion. During coevolution with their hosts, however, some powdery mildews might have acquired additional VLCFA derivatives as signals for accelerated germination, a process that would be receivable, because the wax composition varies between plant species. Our results show the conservation of ancient pathogenicity determinants in modern powdery mildew pathogens more than 140 to 150 million years ago, a time at which the monocot-dicot split is expected to have taken place (Chaw et al., 2004). Modifying the epicuticular wax signature might, therefore, be a promising approach toward plant protection.

MATERIALS AND METHODS

Plant Material and Fungal Infection Assays

The barley (Hordeum vulgare) mutant lwa1 was created by chemical mutagenesis (NaN₃) of the near-isogenic backcross line Ingridmlo5 (obtained from the Max Planck Institute for Plant Breeding Research, Cologne, Germany) as described in Jansen et al., 2007. Barley plants segregating for lwa1 and emr1 alleles were recruited from the cross emr1 × Grannenlose Zweizeiligemlo11 (Grannenlose Zweizeiligemlo11 was obtained from the Max Planck Institute for Plant Breeding Research). Plants homozygous for the wild-type MLO and the 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 µmol m⁻² s⁻¹). Infection assay with Magnaporthe oryzae was done as described in Jansen et al., 2007.

Arabidopsis (Arabidopsis thaliana) plants used in this study were transcriptionally silenced for AtCUT1 because of sense suppression by the transgene 35S:AtCUT1 (Millar et al., 1999) and the respective wild-type accession Col-0. Plants were sown in soil, stratified for 2 d at 4°C in darkness, and grown for 2 weeks in a phytotron (22°C, 60% humidity, and an 8.5-h photoperiod; 120 µmol m⁻² s⁻¹). Thereafter, seedlings were transferred to long-day conditions for 4 weeks (22°C, 50%–60% humidity, and a 18-h photoperiod; 110 µmol m⁻² s⁻¹).

Powdery mildew (Blumeria graminis f. sp. hordei, Bgh) race K1 (Hinze et al., 1991) was provided by Paul Schulze-Lefert (Max Planck Institute for Plant Breeding Research), whereas B. graminis f. sp. tritici (Bgt) was a field isolate collected near Aachen, Germany. Both species were propagated in separate plant cabinets (18°C, 65% humidity, and a 16-h photoperiod; 130–150 µmol m⁻² s⁻¹; Bgh on barley cv IngridMLO and Bgt on wheat cv Feldkrone) by weekly inoculation of 7-d-old seedlings. One day before inoculation, old conidiospores were removed from infected plants by shaking. Adaxial surfaces of 7-d-old primary barley leaves or 6-week-old Arabidopsis plants were inoculated by shaking infected plants over horizontally fixed leaves or detached leaves on kinetin agar (0.8% [w/v] agar-agar, 2.5 µg mL⁻¹ of kinetin) in a settling tower at an average spore density of 1 to 15 conidiospores mm⁻². After settling of conidiospores for 1 h, plants were returned to growth cabinets and cultivated under the conditions described above.

Golovinomyces orontii and Erysiphe pisi f. sp. medicaginis were provided by Ralph Panstruga (Institute for Biology I, Rheinisch-Westfaelische Technische Hochschule [RWTH] Aachen University). G. orontii was maintained on Col-0 plants (20°C, 55%–60% humidity, and an 8-h photoperiod; 100 µmol m⁻² s⁻¹). Ten to eleven days post inoculation, conidiospores from infected plants were used to inoculate test plants by brush inoculation. E. pisi was propagated on susceptible Pisum sativum plants (22°C, 50%–60% humidity, and a 10-h photoperiod; 160 µmol m⁻² s⁻¹). Adaxial surfaces of either 7-d-old primary leaves of barley or 6-week-old Arabidopsis plants were inoculated by shaking infected plants (10 d post inoculation) over them. Inoculated plant material was incubated under 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 µmol m⁻² s⁻¹). For inoculation, 7-d-old barley primary leaves were sprayed with a suspension of uredospores in freon (1,1,2-trichloro-1,2,2-trefluoroethan) as described in Jansen et al., 2007. Inoculated plants were incubated in a dark moist chamber for 24 h and then kept under the same conditions as described above (Supplemental Fig. S8).

Phakopsora pachyrhizi was isolated from infected plant material collected in Brazil and maintained on the soybean (Glycine max) cv Petrina as described in Loehrer et al., 2008. Uredospore suspension for inoculation was generated by washing 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 and placed in a dark moist chamber for 24 h. Thereafter, plants were kept under normal 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 paper moistened with ethanol:acetic acid (3:1, v/v) to avoid displacement of nongerminated conidiospores (Lyngkjaer and Carver, 1999). Fungal structures were 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 Leica DMR microscope equipped with a JVC digital camera KY-F75U 3-DDC, and software DISKUS, version 4.81.1027, was used for image acquisition. Quantification of 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 from the cross between emr1 and Grannenlose Zweizeiligemlo11 described in Jansen et al., 2007. Therefore, water retention was monitored after spraying of 2-week-old plants with water, and in addition, disease severity was measured after inoculation with M. oryzae using a detached leaf assay that is also described in Jansen et al., 2007. Genomic DNA was isolated from each of these F2 individuals using 30 to 50 mg of leaf material harvested from 2-week-old seedlings. The material was homogenized using a TissueLyser II (Qiagen), and extraction was carried out using the BioSprint DNA Plant Kit and the BioSprint 96 workstation from the same supplier. Isolated DNA was dissolved in distilled water, and, based on agarose gel electrophoresis, DNA concentration of all samples was adjusted to approximately 25 ng µL⁻¹.

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

Construction of the Transformation Vector and Generation of Transgenic Plants

The genomic HvKCS6 sequences of Ingridmlo5 and lwa1 mutant plants were amplified with forward primer 5′-TAGGATCCCAGCACCGTGAACGCATAC-3′ containing a BamHI restriction site and reverse primer 5′-ATGAATTCCTGGTTTAATTTGGTCGGGGC-3′ containing an EcoRI restriction site and cloned into intermediate vector pUBI-ABM (DNA Cloning Service) between the maize (Zea mays) UBIQUITIN-1 (UBI1) promoter with first intron and the NOPALINE SYNTHASE termination sequence of Agrobacterium tumefaciens. Correct clones were verified by sequencing, and the whole expression cassette was subcloned into binary vector p6int-UBI (DNA Cloning Service) through SfiI restriction sites. The binary vector was introduced into A. tumefaciens strain AGL-1 by electroporation as described elsewhere (Hensel et al., 2009).

Stable transgenic barley plants were obtained by Agrobacterium spp.-mediated gene transfer following a method described previously (Hensel et al., 2009) with slight modifications. Briefly, immature embryos of both genotypes were precultured for 5 d on blast cell induction medium with dicamba at a final concentration of 5 mg L⁻¹ at 24°C in the dark (Hensel et al., 2009). After agroinfection, precultured immature embryos were cocultured at stacks on moistened filter paper (300 µL of blast cell conditioned medium; Hensel et al., 2009) in 5.5-cm diameter petri dishes for 2 d in the dark. Callus induction was performed on blast cell induction medium with either 20 or 50 mg L⁻¹ of hygromycin B (Roche Diagnostics). Regeneration took place on blast cell recovery medium with 25 mg L⁻¹ of hygromycin B at 24°C in the light (Hensel et al., 2009). In contrast to the previously published method, all regenerants were further processed, excepting that some of the primary transgenic plants may be derived from the same transformation event. Rooting and transfer into soil were the same as described before.

Transgenic events were identified and confirmed by PCR based on primer pairs specific for the junction between ZmUBI-1:hygromycin phosphotransferase (5′-TTTAGCCCTGCCTTCATACG-3′ and 5′-GATTCCTTGCGGTCCGAATG-3′) and ZmUBI-1:HvKCS6 (5′-TTTAGCCCTGCCTTCATACG-3′ and 5′-TGCACCAGCCTGTACTTGG-3′) using 100 ng of genomic DNA isolated following the protocol in Pallotta et al., 2000. To identify siblings, DNA gel-blot analyses were conducted using an hygromycin phosphotransferase-specific hybridization probe that was constructed with the same primers as for PCR and a DIG-PCR Labeling Kit (Roche Diagnostics). Genomic DNA (30 µg) were digested with HindIII and separated by agarose gel electrophoresis. After transfer on a positively charged nylon membrane (Roche Diagnostics), blots were hybridized and processed following 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 fourth leaf of each individual 5-week-old plant was used for wax analysis. The corresponding leaf area was determined by scanning of dipped leaves and pixel quantification using Adobe Photoshop CS2, version 9.0. The resulting solution containing the cuticular waxes was spiked with 10 µg of tetracosane (Sigma-Aldrich) as an internal standard. The solvent was evaporated under a stream of nitrogen, and compounds containing free hydroxyl and carboxyl groups were converted into their trimethylsilyl ethers and esters, respectively, with bis-(N,N-trimethylsilyl)-trifluoroacetamide (Machery-Nagel) in pyridine for 40 min at 70°C before GC-MS analysis. Wax constituents were identified by their electron impact MS spectra after GC-MS analysis and quantified using an identical GC system equipped with a flame ionization detector (FID) as described previously (Richardson et al., 2005).

Phylogenetic Analysis

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

Sequence data from this article can be found in the EMBL/GenBank data libraries or the Arabidopsis Genome Initiative under the following accession numbers: 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 Arabidopsis KCS sequences used for phylogenetic analysis are provided in Supplemental Figure S6.

Supplemental Data

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

• Supplemental Figure S1. Powdery mildew infection structures on barley and Arabidopsis.

• Supplemental Figure S2. Detailed analysis of cuticular wax profiles from different barley genotypes.

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

• Supplemental Figure S4. Southern-blot confirmation of transfer-DNA integration pattern.

• Supplemental Figure S5. Microscopic analysis of postpenetration developmental stages of Blumeria graminis f. sp. hordei.

• Supplemental Figure S6. Phylogenetic tree of barley and Arabidopsis KCS protein families.

• Supplemental Figure S7. Microscopic analysis of the development of Golovinomyces orontii on Arabidopsis transgenic plants (35S:AtCUT1) with sense silencing of AtCUT1.

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

• Supplemental Table S1. Genotype data for 21 informative SNPs from chromosome 4H and emr1 and lwa1 loci across 92 F2 offspring originating from an emr1 × Grannenlose Zweizeiligemlo11 cross.

Glossary

Col-0

Columbia-0

FAE

fatty acid elongase

FID

flame ionization detector

GC

gas chromatography

hpi

h post inoculation

MS

mass spectrometry

SEM

scanning electron microscopy

SNP

single-nucleotide polymorphism

VLCFA

very long-chain fatty acid