The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

Discovery of genes affecting resistance of barley to adapted and non-adapted powdery mildew fungi.

BACKGROUND: Non-host resistance, NHR, to non-adapted pathogens and quantitative host resistance, QR, confer durable protection to plants and are important for securing yield in a longer perspective. However, a more targeted exploitation of the trait usually possessing a complex mode of inheritance by many quantitative trait loci, QTLs, will require a better understanding of the most important genes and alleles. RESULTS: Here we present results from a transient-induced gene silencing, TIGS, approach of candidate genes for NHR and QR in barley against the powdery mildew fungus Blumeria graminis. Genes were selected based on transcript regulation, multigene-family membership or genetic map position. Out of 1,144 tested RNAi-target genes, 96 significantly affected resistance to the non-adapted wheat- or the compatible barley powdery mildew fungus, with an overlap of four genes. TIGS results for QR were combined with transcript regulation data, allele-trait associations, QTL co-localization and copy number variation resulting in a meta-dataset of 51 strong candidate genes with convergent evidence for a role in QR. CONCLUSIONS: This study represents an initial, functional inventory of approximately 3% of the barley transcriptome for a role in NHR or QR against the powdery mildew pathogen. The discovered candidate genes support the idea that QR in this Triticeae host is primarily based on pathogen-associated molecular pattern-triggered immunity, which is compromised by effector molecules produced by the compatible pathogen. The overlap of four genes with significant TIGS effects both in the NHR and QR screens also indicates shared components for both forms of durable pathogen resistance.

Convergent evidence for a role of WIR1 proteins during the interaction of barley with the powdery mildew fungus Blumeria graminis.

Pathogen attack triggers a multifaceted defence response in plants that includes the accumulation of pathogenesis-related proteins and their corresponding transcripts. One of these transcripts encodes for WIR1, a small glycine- and proline-rich protein of unknown function that appears to be specific to grass species. Here we describe members of the HvWIR1 multigene family of barley with respect to phylogenetic relationship, transcript regulation, co-localization with quantitative trait loci for resistance to the barley powdery mildew fungus Blumeria graminis (DC.) E.O. Speer f.sp. hordei, the association of single nucleotide polymorphisms or gene haplotypes with resistance, as well as phenotypic effects of gene silencing by RNAi. HvWIR1 is encoded by a multigene family of moderate complexity that splits up into two major clades, one of those being also represented by previously described cDNA sequences from wheat. All analysed WIR1 transcripts accumulated in response to powdery mildew attack in leaves and all mapped WIR1 genes were associated with quantitative trait loci for resistance to B. graminis. Moreover, single nucleotide polymorphisms or haplotypes of WIR1 members were associated with quantitative resistance of barley to B. graminis, and transient WIR1 gene silencing affected the interaction of epidermal cells with the pathogen. The presented data provide convergent evidence for a role of the HvWIR1a gene and possibly other family members, during the interaction of barley with B. graminis.

Basal host resistance of barley to powdery mildew: connecting quantitative trait Loci and candidate genes.

The basal resistance of barley to powdery mildew (Blumeria graminis f. sp. hordei) is a quantitatively inherited trait that is based on nonhypersensitive mechanisms of defense. A functional genomic approach indicates that many plant candidate genes are involved in the defense against formation of fungal haustoria. It is not known which of these candidate genes have allelic variation that contributes to the natural variation in powdery mildew resistance, because many of them may be highly conserved within the barley species and may act downstream of the basal resistance reaction. Twenty-two expressed sequence tag or cDNA clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcriptional profiling, gene silencing, or overexpression data, as well as mlo, Ror1, and Ror2, were mapped and considered candidate genes for contribution to basal resistance. We mapped the quantitative trait loci (QTL) for powdery mildew resistance in six mapping populations of barley at seedling and adult plant stages and developed an improved high-density integrated genetic map containing 6,990 markers for comparing QTL and candidate gene positions over mapping populations. We mapped 12 QTL at seedling stage and 13 QTL at adult plant stage, of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTL identified for basal resistance to powdery mildew. This co-localization justifies giving priority to those six candidate genes to validate them as being responsible for the phenotypic effects of the QTL for basal resistance.

A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance.

Higher plants possess large multigene families encoding secreted class III peroxidase (Prx) proteins. In barley, two Prx cDNAs encoding HvPrx07 and HvPrx08 have been isolated and characterized to some extent with respect to a resistance-mediating function upon attack by the powdery-mildew fungus Blumeria graminis f.sp. hordei (Bgh). Here we present evidence for the tissue-specific accumulation of a new Prx mRNA, HvPrx40, in Bgh-attacked epidermis of barley (Hordeum vulgare). The encoded protein is predicted to be secreted into the apoplastic space of epidermal cells due to the absence of a C-terminal extension, which distinguishes it from other Prx proteins reported to accumulate in leaf epidermis. Transient overexpression of HvPrx40 enhanced the resistance of wheat (Triticum aestivum) and barley against Blumeria graminis f.sp. tritici (wheat powdery mildew) and Bgh, respectively. These findings were complemented by transient-induced gene silencing showing hypersusceptibility of barley leaf epidermal cells to Bgh. The local accumulation of oxidized 3,3-diaminobenzidine that reflects H2O2 production at sites of attempted fungal penetration was not reduced in HvPrx40-silenced cells, suggesting a role of this peroxidase other than the production of reactive oxygen species.