Identification of novel genetic factors underlying the host-pathogen interaction between barley (Hordeum vulgare L.) and powdery mildew (Blumeria graminis f. sp. hordei)

Abstract

Powdery mildew is an important foliar disease of barley (Hordeum vulgare L.) caused by the biotrophic fungus Blumeria graminis f. sp. hordei (Bgh). The understanding of the resistance mechanism is essential for future resistance breeding. In particular, the identification of race-nonspecific resistance genes is important because of their regarded durability and broad-spectrum activity. We assessed the severity of powdery mildew infection on detached seedling leaves of 267 barley accessions using two poly-virulent isolates and performed a genome-wide association study exploiting 201 of these accessions. Two-hundred and fourteen markers, located on six barley chromosomes are associated with potential race-nonspecific Bgh resistance or susceptibility. Initial steps for the functional validation of four promising candidates were performed based on phenotype and transcription data. Specific candidate alleles were analyzed via transient gene silencing as well as transient overexpression. Microarray data of the four selected candidates indicate differential regulation of the transcription in response to Bgh infection. Based on our results, all four candidate genes seem to be involved in the responses to powdery mildew attack. In particular, the transient overexpression of specific alleles of two candidate genes, a potential arabinogalactan protein and the barley homolog of Arabidopsis thaliana's Light-Response Bric-a-Brac/-Tramtrack/-Broad Complex/-POxvirus and Zinc finger (AtLRB1) or AtLRB2, were top candidates of novel powdery mildew susceptibility genes.

Fig 1. Biplot of the phenotypic distribution of the 267 Hordeum vulgare accessions included in the phenotyping-panel.

Phenotypic values correspond to the arithmetic mean (n ≥3) of the normalized infected leaf area [in %] for the Blumeria graminis f. sp. hordei isolates D35/3 vs. RiIII and the corresponding absolute frequencies of genotypes per interval and isolate.

Fig 2. Hierarchical cluster analysis of the 201 Hordeum vulgare accessions of the GWA-panel based on the Rogers‘ distance.

The identified subpopulation of 28 genotypes is highlighted in blue, modern material is labeled with red arrow-heads and carriers of the resistant alleles are indicated with colored dots: green–CG_1; ochre–CG_2; violet–CG_3; blue–CG_4.

Fig 3. Manhattan plots of the–log₁₀-transformed p-values for Max trait, RiIII, and D35/3 severities.

The significance threshold of -log₁₀ (p-value) = 4.45 is depicted as a pink dotted line and the blue arrows represent the peaks from which the four candidate genes have been selected. The 17 SNPs, which were significantly associated with all three traits, are indicated as red diamonds.

Fig 4. Relative expression of the four Candidate Genes (CG) in powdery mildew attacked epidermis cells.

The quantile-normalized signal intensities represent the relative expression of each CG based on Microarray data [68] for non-inoculated, Blumeria graminis f. sp. hordei (isolate CH4.8) or Blumeria graminis f. sp. tritici (isolate FAL 92315) inoculated epidermal peels of the resistant barley cultivar Vada for the indicated time points post-inoculation. The data embody arithmetic means (n = 3) plus standard error of the mean. * p-value < 0.05; ** p-value < 0.01.

Fig 5. Heatmap-plot of the linkage disequilibrium levels as r² values for the Single-Nucleotide Polymorphisms (SNPs) of the selected Candidate Genes (CG).

The corresponding p-values for each r² value were < 0.0001 based on the two-sided Fisher’s exact test.

Fig 6. Box plots of the log₂-transformed relative susceptibility indices of transiently transformed Hordeum vulgare epidermis cells.

The error bars represent the minimal and maximal values of the susceptibility indices relative to the empty vector control for the transient silencing (n = 5) (A) of the indicated candidates (CG) and for the transient overexpression (n = 4) (B) in a resistant (WB-052, hollow boxes) and a susceptible barley genotype (Morex, gray-shaded boxes) after inoculation with the Blumeria graminis f. sp. hordei isolate CH4.8, whereas the putative resistant (res) and the putative susceptible (sus) alleles were tested in the transient overexpression. The corresponding positive controls for the transient silencing: the barley Mlo gene (pIPKTA36) and for the transient overexpression: a wheat Class III peroxidase (Prx, pJP01) are depicted [43,69]. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.