Phytoalexin Gene Regulatory Networks

植物抗毒素是一种防御代谢物，是植物在识别病原体衍生的激发子分子后合成的。尽管转录因子（TF）蛋白在保护作物免受病原体侵害中起着关键作用，但它们对植物抗毒素基因表达的调控仍知之甚少。我们最近发现TF蛋白GmNAC42-1和GmMYB29A2激活大豆主要植物抗毒素glyceollins的生物合成。然而，在缺乏激发子的情况下，单独过表达每个TF不足以激活整个甘油生物合成途径。这表明需要其他tf。出乎意料的是，我们发现GmNAC42-1和GmMYB29A2分别是拟南芥和葡萄中吲哚生物碱和stilbene TF基因的同源物。因此，在植物中可能存在一组保守的TFs来调节多种植物抗毒素途径。目标-我们研究计划的长期目标是了解植物抗毒素基因调控的机制和进化。我们的短期目标是：1)确定缺失的调节甘油生物合成的TF；2)确定植物抗菌素生物合成基因是否共同进化出GmNAC42和GmMYB29A2识别的DNA元件。如果这是真的，后者将表明GmNAC42-和gmmyb29a2型tf在很大程度上负责将多种代谢基因纳入植物抗毒素的生物合成中。为了解决第一个目标，我们将确定核dna -蛋白复合物（即染色质）中哪些TF蛋白与GmNAC42-1和GmMYB29A2共定位。然后，我们将在诱导的大豆毛状根中过表达和沉默这些基因，以测试它们是否在调节甘油生物合成中起作用。我们将对在之前的转录组学实验中被鉴定为与GmNAC42-1和GmMYB29A2共调控的TF基因做同样的研究。为了实现第二个目标，我们将通过染色质免疫沉淀-下一代测序（ChIP-seq），然后通过高通量启动子报告子和酵母单杂交（Y1H）测定，确定与GmNAC42和/或GmMYB29A2结合的共识DNA元件。最后，我们将确定来自一个植物物种的GmNAC42-1和GmMYB29A2基因是否可以弥补其他植物物种的功能缺失突变。鉴定缺失的甘油转录因子将为区分植物抗毒素基因调控网络（GRN）的保守成分和物种特异性成分提供基础。确定基因是否通过其TF识别元件被纳入病原体诱导调节，将有助于了解植物抗毒素grn是如何进化的。我们的研究将为学生提供多学科的培训机会。最后，研究的重点是了解甘油作为模型的调控，因为它们对大豆抵御大豆疫霉至关重要，大豆疫霉是大豆的第二大破坏性病原体，每年给加拿大造成5000万美元的产量损失。

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.