Phytoalexin Gene Regulatory Networks

植物抗毒素是一种防御代谢产物，在植物中识别病原体衍生的分子（称为激发子）后合成。转录因子（TF）蛋白对植物抗毒素基因表达的调控仍然知之甚少，尽管它们在保护作物免受病原体侵害方面发挥着关键作用。我们最近发现TF蛋白GmNAC 42 -1和GmMYB 29 A2激活大豆抗毒素（大豆的主要植物抗毒素）的生物合成。然而，在没有激发子的情况下单独过表达每种TF不足以激活整个大豆抗毒素生物合成途径。这表明需要其他的工作队。出乎意料的是，我们发现GmNAC 42 -1和GmMYB 29 A2分别是拟南芥和葡萄藤中吲哚生物碱和二苯乙烯TF基因的同源物。因此，植物中可能存在一组保守的转录因子，调节多种植物抗毒素途径。 目的了解植物抗毒素基因调控的机制和进化是本研究计划的长期目标。我们的短期目标是：1）鉴定调节大豆抗毒素生物合成的缺失TF;和2）确定植物抗毒素生物合成基因是否具有通常进化的被GmNAC 42和GmMYB 29 A2识别的DNA元件。如果是后者，则表明GmNAC 42和GmMYB 29 A2型转录因子在很大程度上负责将不同的代谢基因引入植物抗毒素的生物合成。 为了解决第一个目标，我们将确定哪些TF蛋白与GmNAC 42 -1和GmMYB 29 A2在核DNA-蛋白质复合物（即染色质）中共定位。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大豆抗毒素生物合成中发挥作用。我们将对在我们先前的转录组学实验中鉴定为与GmNAC 42 -1和GmMYB 29 A2共调节的TF基因进行相同的操作。 为了解决第二个目标，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-报告基因和酵母单杂交（Y1 H）测定来鉴定GmNAC 42和/或GmMYB 29 A2结合的共有DNA元件。最后，我们将确定来自一种植物物种的GmNAC 42 -1和GmMYB 29 A2基因是否可以补充其他植物物种的功能丧失突变。 鉴定缺失的大豆抗毒素转录因子将为区分植物抗毒素基因调控网络（GRN）的保守组分与物种特异性组分提供基础。确定基因是否被它们的TF识别元件吸收到病原体诱导的调节中，将提供对植物抗毒素GRNs如何进化的理解。我们的研究将为学生提供多学科的培训机会。最后，研究重点是了解大豆抗毒素作为模型的调节，因为它们对于大豆防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，每年使加拿大损失5000万美元。

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.