Phytoalexin Gene Regulatory Networks

植保素是一种防御代谢物，在植物中被称为激发子的病原体衍生分子识别后合成。尽管转录因子(TF)蛋白在作物抵御病原菌中起着重要作用，但对转录因子(TF)蛋白对植物抗病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激活了大豆主要植物保卫素--甘油三酯的生物合成。然而，在没有激发子的情况下，单独过度表达每个转铁蛋白并不足以激活整个糖蛋白生物合成途径。这表明还需要其他的信托基金。出人意料的是，我们发现GmNAC42-1和GmMYB29A2分别是拟南芥和葡萄中吲哚生物碱和二苯乙烯类TF基因的同源物。因此，可能存在一组保守的转录因子，调节植物中不同的植物保卫素途径。 目的我们的研究计划的长期目标是了解植物保卫素基因调控的机制和进化。我们的短期目标是：1)确定调控甘油三酯生物合成的缺失的TF(S)；2)确定植物保卫素生物合成基因是否共同进化出被GmNAC42和GmMYB29A2识别的DNA元件。如果是真的，后者将表明GmNAC42-和GmMYB29A2类型的转录因子在很大程度上负责将不同的代谢基因参与植物保卫素的生物合成。 为了解决第一个目标，我们将确定Tf蛋白与GmNAC42-1和GmMYB29A2在核DNA-蛋白质复合体(即染色质)中共定位的内容。然后，我们将在诱导的大豆毛状根中过度表达和沉默这些基因，以测试它们是否在调节甘油三酯生物合成中发挥作用。我们将对在先前的转录学实验中被鉴定为与GmNAC42-1和GmMYB29A2共调控的TF基因进行同样的处理。 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)以及高通量启动子-报告程序和酵母单杂交(Y1H)分析来鉴定与GmNAC42和/或GmMYB29A2结合的共识DNA元件。最后，我们将确定一个植物物种的GmNAC42-1和GmMYB29A2基因是否可以补充其他植物物种的功能丧失突变。 鉴定缺失的糖蛋白转录因子将为区分植物保卫素基因调控网络(GRN)的保守成分和物种特异性成分提供基础。确定基因是否被其Tf识别元件增选到病原体诱导的调控中，将有助于理解植物抗毒素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，这项研究的重点是了解甘油三酯作为模型的调节，因为它们对于大豆对大豆疫霉的防御至关重要。大豆疫霉是大豆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.