Phytoalexin Gene Regulatory Networks

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Phytoalexins are defense metabolites that are synthesized in plants upon recognition of pathogen-derived molecules called elicitors. The regulation of phytoalexin gene expressions by transcription factor (TF) proteins remains poorly understood despite their critical roles in defending crops against pathogens. We recently discovered that the TF proteins GmNAC42-1 and GmMYB29A2 activate the biosynthesis of glyceollins, soybean's major phytoalexins. Yet, overexpressing each TF alone in the absence of an elicitor was insufficient to activate the entire glyceollin biosynthetic pathway. This suggested that other TFs were required. Unexpectedly, we found that GmNAC42-1 and GmMYB29A2 were homologs of indole alkaloid and stilbene TF genes from Arabidopsis and grapevine, respectively. Thus, there may exist a conserved group of TFs that regulates diverse phytoalexin pathways in plants. Objectives - The long-term goals of our research program are to understand the mechanism and evolution of phytoalexin gene regulation. Our short-term objectives are: 1) to identify the missing TF(s) that regulate glyceollin biosynthesis; and 2) to determine whether phytoalexin biosynthesis genes have commonly evolved DNA elements that are recognized by GmNAC42 and GmMYB29A2. If true, the latter would suggest that GmNAC42- and GmMYB29A2-type TFs are largely responsible for coopting diverse metabolism genes into phytoalexin biosynthesis. To address the first aim we will identify what TF proteins co-localize with GmNAC42-1 and GmMYB29A2 in nuclear DNA-protein complexes (i.e. chromatin). We will then overexpress and silence these in elicited soybean hairy roots to test whether they have roles in regulating glyceollin biosynthesis. We will do the same for the TF genes that were identified as being coregulated with GmNAC42-1 and GmMYB29A2 in our prior transcriptomics experiments. To address the second aim, we will identify the consensus DNA elements that are bound by GmNAC42 and/or GmMYB29A2 by chromatin immunoprecipitation-next generation sequencing (ChIP-seq) followed by high-throughput promoter-reporter and yeast one-hybrid (Y1H) assays. Finally, we will determine whether GmNAC42-1 and GmMYB29A2 genes from one plant species can complement the loss-of-function mutations of other plant species. Identifying the missing glyceollin TFs will provide a basis for distinguishing conserved versus species-specific components of the phytoalexin gene regulatory network (GRN). Determining whether genes were coopted into pathogen-inducible regulation by their TF recognition elements will provide an understanding of how phytoalexin GRNs evolved. Our research will provide multidisciplinary training opportunities for students. Finally, the research focuses on understanding the regulation of glyceollins as a model since they are critical for soybean's defense against Phytophthora sojae, the second most destructive pathogen of soybean that costs Canada 50 million dollars in yield loss annually.