The Role Of CHH Methylation In Wheat Powdery Mildew Defense

The powdery mildew disease caused by Blumeria graminis tritici (Bgt) inflicts severe economic losses in worldwide wheat crops. Systematic understanding of gene interactions and molecular mechanisms involved in wheat resistance to Bgt is essential. Aegilops tauschii is considered the D genome progenitor of hexaploid wheat and thus an essential resource for wheat resistance. DNA methylation is a critical epigenetic mark that plays a key role in regulating gene expression and is involved in plant defense. Determination of the epigenetic mechanisms in Aegilops tauschii leaves during the susceptible interaction with Bgt should provide insights into future research directions for crop defense and breeding. In this report, we studied the effect of epigenetic changes and their effects on plant defense. The major findings are summarized below:We found that 12 h after infection (hai) is a critical time point after which resistant and susceptible lines start to diverge in response to Bgt infection. This was determined by using the PM resistant accession Y2280 of Aegilops tauschii (Y2280) and PM susceptible line AL8/78.We then profiled RNA expression patterns and AGO4a pull-down sRNAs in Aegilops tauschii leaves after 12 h susceptible interaction with Bgt using Illumina deep sequencing technology. We found that AGO4a preferentially binds to 24 nt small RNAs with 5 A. Compared with the control, the number of AGO4a pull-down 24-nt sRNAs was significantly reduced after 12 hai. The effect was stonger in the resistant line than that of the susceptible line, suggesting that powdery mildew reduced the number of 24 nt sRNAs captured by AGO4a.We next generated whole-genome cytosine methylation maps at single-nucleotide resolution. We also studied RNA expression levels in AL8/78 leaves at 12 hai using Illumina deep sequencing. We identified 34 hyper-methylated regions and 104 hypo-methylated regions in CG context,26 hyper-methylated regions and 106 hypo-methylated regions in CHG context,1196 hyper methylated regions and 13673 hypo-methylated regions in CHH context in the inoculated leaves compared with those of the noninfected control. We classified the hypo-methylated regions associated genes in CHH context into different groups by molecular function using the Gene Ontology (GO) categorization from the AgriGO database. The results showed that molecular function groups corresponding to stimulus and lipid metabolic process were significantly enriched among differentially methylated genes, suggesting that powdery mildew, via CHH methylation inhibition, induced gene expressions responding to stimulus and lipid metabolic processes.Our analysis identified that CHH hypo-methylation of Circadian Clock Associated1 (CCA1) gene was significantly reduced after 12 hai. In addition, under continuous light conditions, powdery mildew did not compromise the circadian clock but rather led to its reinforcement, suggesting that powdery mildew via circadian clock CHH methylation inhibition, induced the circadian clock reinforcement to gate immune response.We further identified that the Peroxidase genes (POX5 and POX22), Cysteine-rich repeat secretory protein 55 precursor (CRP55), Cysteine-rich receptor-like protein kinase (CRP10 and CRK25), hormone metabolism brassinosteroid synthesis-degradation sterols (CYP51), and Methyl Jasmonate (JA) pathway Lipoxygenase (LOX) are CHH hypo- methylated. Accrodingly, mRNA level of CHH hypo-methylation genes, as detected by quantitative real-time-PCR (q-PCR) in Bgt-treated plants at 0,6,12, and 24 hai, were mainly increased. Transient overexpression assay of the POX5 gene in Aegilops tauschii epidermal cells showed enhancement to Bgt resistance.Together, this study provides useful information on the role of DNA methylation during powdery mildew infection which may involve AGO4a-sorted 24 nt siRNAs through circadian clock reinforcement to gate immune response.