Annotation, phylogenetic analysis and functional characterization of barley [Hordeum vulgare L] WRKY transcription factors in the interaction with powdery mildew fungus Blumeria graminis
WRKY transcription factors are a conserved superfamily distributed in eukaryotes and extremely expanded in flowering plants. They are central regulators of diverse plant cellular responses such as biotic and abiotic stresses. Despite the economic importance of the crop plant barley (Hordeum vulgare L.) and the significance of WRKY transcriptional network, the barley WRKY transcription factors are largely understudied and undetermined. The present work aimed to perform a whole-genome gene discovery of barley WRKY family and functionally characterize two of the important genes HvWRKY1 and HvWRKY2 in the interaction with barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh).Based on the NCBI databases and the draft sequence of barley genome, blastp, blastn and tblastn searches were performed to find putative barley WRKY sequences. As a result, 100 unique WRKY members containing at least one WRKY domain were found in the barley genome. Apart from the previously annotated 45 HvWRKY members, the novel sequences were designated HvWRKY47-HvWRKY102. Expansion of group III members was found in barley compared with the model plant Arabidopsis thaliana. Within the group III members, WRKYs with a non-canonical WRKYGEK motif were proven to be a monocot-specific group and this diversification implicates special functions in monocot plants. Using artificial microRNA-based transient silencing of HvWRKY2, I observed on single-cell level enhanced resistance to Bgh. Together with previous findings this result confirmed its nature as negative regulator in barley-Bgh interaction. Whereas the R-protein Mla10 was identified as an upstream factor for HvWRKY1 and HvWRKY2, downstream target promoters are unknown for barley WRKYs. HvGER4c is one of the most abundant pathogenesis-related (PR) genes induced in Bgh-infected barley leaf epidermal cells and contains enriched WRKY-binding sites in the promoter. The wild-type and W-box mutated versions of HvGER4c promoter GUS fusion constructs were used in the co-bombardment assay with 35S::HvWRKY1/2 plasmids to verify their possible interaction. Co-expression of 35S::HvWRKY1/2 constructs with pHvGER4c::GUS significantly and specifically suppressed the Bgh-induced activity of HvGER4c promoter, with HvWRKY2 showing stronger repression activity than HvWRKY1. The finding indicates a negative transcriptional regulation of HvWRKY1/2 on the defense-related gene HvGER4c. In addition, it was observed that the HvWRKY2-mediated repression of HvGER4c was independent of Mla12 or Mlo, which were previously identified as an effector-dependent interaction partner of HvWRKY1/2, or assumed to be required for HvWRKY2-mediated compatibility respectively. Moreover, electrophoretic mobility shift assay (EMSA) was used to analyze the binding of HvWRKY2 recombinant protein with W-box elements in HvGER4c promoter. HvWRKY2 protein showed binding affinity to all the tested W-boxes but with questionable specificity. Transient overexpression of the Arabidopsis homologue AtWRKY40 resulted in enhanced susceptibility to Bgh in barley. The retained activity of AtWRKY40 between dicot and monocot indicated evolutionary conserved functions of IIa WRKY factors as negative regulators in basal defense. Taken together, HvWRKY1 and HvWRKY2 act as negative regulators largely dependent on their transcriptional repression of the downstream target genes, including the verified HvGER4c gene. Some further candidate WRKY genes involved in plant immune responses were suggested based on the microarray expression profile after pathogen challenge. Whole-genome annotation and phylogenetic analysis of barley WRKY transcription factors might provide insights into further characterization and cross-species comparison of the conserved family.
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