组织特异侵染过程中大豆疫霉效应子的转录调控机制与功能解析

Many Phytophthora pathogens can cause plant diseases by colonizing different organs of host. However, the components, metabolisms, and defense responses vary significantly in different plant organs, and how these organisms regulate to infect different organs is largely unknown. Phytophthora sojae is the agent of soybean stem and root rot disease, and is able to infect roots and leaves of soybean. Between the specific infection of roots and leaves, we recently found transcription levels of many P. sojae effector genes were significant different. Based on CRISPR/Cas9-mediaed gene knock-out, we also identified a bZIP transcription factor of P. sojae that might regulate specific infection of roots. These data have preliminarily revealed that transcriptional regulation may play an important role for organ-specific infection of P. sojae. In this project, we plan to: (1) compare the transcriptomes of gene knock-out and over-expressed mutants of the bZIP transcription factor, and predict its regulated effector genes; (2) validate protein-DNA interaction between the transcription factor proteins and effector promoter DNAs, and reveal associated transcriptional regulation mechanism; (3) knock out the effector genes that are regulated by the transcription factor, and learn effector gene functions for organ-specific infection of P. sojae. These studies may identify organ-specific infection-related effectors and revealed the associated transcriptional regulation network in P. sojae. The results may provide candidate targets for design of new fungicide for Phytophthora disease control, and provide a theoretical basis for the disease strategy on interference of pathogenesis-related transcriptional regulation.

许多疫霉菌能够侵染寄主植物的不同组织，然而植物的不同组织在构成、代谢及抗病等方面存在显著差异，目前对于疫霉菌如何调节以适应组织特异侵染的机制了解甚少。前期我们通过比较大豆疫霉侵染大豆根和叶的转录组数据发现，许多效应子的转录水平存在明显差异，同时还通过CRISPR介导的基因敲除体系鉴定到一个与根部侵染特异相关的转录因子，初步揭示了转录调控对于疫霉菌的组织特异侵染至关重要。本项目拟进一步分析比较该转录因子的基因敲除和过表达转化子的转录组，筛选可能受其调控的组织特异侵染相关的效应子，并验证转录因子蛋白与效应子启动子DNA元件之间的特异性互作，进而系统解析这些效应子的生物学功能。期望鉴定出大豆疫霉特异侵染大豆不同组织的关键效应子并绘制相关的转录因子对效应子的调控网络，为疫霉菌防控新药剂的开发提供候选靶标，也为利用寄主诱导基因沉默(HIGS)技术干扰病菌致病过程转录调控的病害防控策略提供理论依据。

许多病原菌能够侵染植物的不同组织，而植物的不同组织在结构构成、代谢与抗病反应等方面均存在显著差异，那么病原菌是如何调节自身不同致病因子以侵染不同组织的呢？本研究聚焦大豆疫霉菌对大豆根和叶的特异侵染，发现细胞壁降解酶、转运蛋白、氧化还原相关酶类以及效应子在内的分泌蛋白等相关基因受到了显著的差异调控，其中PsBZP29是大豆疫霉侵染大豆根部时特异表达且显著影响致病力的关键bZIP转录因子。通过敲除或过表达侵染根部或叶部时特异表达且受PsBZP29转录调控的效应子基因，发现种内特异进化（且数量扩张）的Group 2角质酶编码基因特异参与叶部侵染，而PsRSE1是根部特异侵染相关的RxLR效应子。为研究效应子的转录调控机制，在利用ATAC-seq分析全基因组开放染色质进而明确高可信度顺式作用元件的基础上，以效应子启动子区普遍存在的M93元件为探针，通过筛选酵母单杂交文库和互作验证，鉴定了与之结合的Myb转录因子PsMyb37；经基因敲除与回补明确了PsMyb37为大豆疫霉的致病关键调控因子，PsMyb37通过结合启动子的M93元件影响多个效应子在侵染早期的上调表达。研究结果促进了对大豆疫霉致病机理及转录因子介导的转录调控机制的深入理解，同时也发展了对疫霉菌转录调控研究的技术体系。相关结果已在《Molecular Plant Pathology》《Frontiers in Genetics》《植物病理学报》发表第一标注论文3篇，在《PLoS Pathogens》《Molecular Plant Pathology》《Frontiers in Microbiology》等发表第二标注论文7篇，另有其它有标注的论文5篇；与本项目研究相关的5名研究生和3名本科生等均已顺利毕业。