辣椒疫霉P4-ATP酶PcAPT1介导的蛋白外泌及其在致病过程中的作用机制研究

Phytophthora blight, caused by Phytophthora spp., is one of the most devastating diseases in the world, and the knowledge of molecular mechanisms for pathogenesis of Phytophthora is the basis for disease control. In model eukaryotic cells, P4-ATPase has been shown to mediate phospholipid transfer to regulate vesicle formation and protein secretion in order to maintain normal growth and development of cells..In our preliminary work, we found that P4-type ATPase-encoding gene, PcAPT1, in the economically important oomycetes pathogen Phytophthora capsici, which is required for both foliar and stem infection by Phytophthora capsici. △PcAPT1 mutants are impaired in the secretion of a range of extracellular enzymes. PcAPT1 complement the growth phenotype of a △drs2 mutant, indicating that in yeast PcAPT1 is able to fulfill the functions of the Drs2, which provide the groundwork for the function of P4-ATPase in regulation pathogenicity of Phytophthora capsici..In this project, in order to further elucidate the regulation mechanism of P4-ATPase on the roles of proteins secretion and the pathogenesis of Phytophthora capsici, the following studies will be conducted. Firstly, we will clarify the biological functions of PcAPT1 in the process of growth, development, and virulence in Phytophthora capsici based on the phenotype analysis of △PcAPT1 mutants. Secondly, we will combine subcellular localization, vesicle formation and phospholipids transport capacity of membrane to explain the regulation mechanisms of PcAPT1 in membrane traffic. Thirdly, yeast two-hybrid analysis, bimolecular fluorescence complementation(BiFC), GST Pulldown and Co-immunoprecipitation(CoIP) assay will be carried out to screen the interaction proteins and explore protein interaction mechanism. Finally, we will clarify the regulation of PcAPT1 on secretion of effector proteins and its function in pathogenesis based on the secreted proteome and the secretion of effector proteins. We expect that these results will help in-depth for our understanding the function of P4-ATPase in Phytophthora capsici, at the same time, it will provide scientific basis for development of novel strategies for control of Phytophthora blight.

由疫霉菌侵染引起的作物疫病是农业生产上的重要病害，从分子水平上认识疫霉菌的致病机制是疫病控制的基础。模式生物P4-ATP酶通过调控囊泡形成及蛋白泌出，从而维持正常生命过程。我们前期研究发现，辣椒疫霉P4-ATP酶基因PcAPT1敲除突变体致病力显著减弱，胞外酶泌出缺陷，且能在酵母缺失突变体中回复功能，证实其具有P4-ATP酶功能。为深入揭示其调控蛋白外泌机制及在致病中的作用，本项目拟进一步：①通过突变体表型分析，明确PcAPT1在辣椒疫霉生长发育和致病过程中的生物学功能；②通过亚细胞定位、囊泡形成及转运能力分析，明确PcAPT1在膜运输中的功能；③应用酵母双杂交和BiFC鉴定互作蛋白及互作机制；④通过外泌蛋白组及对效应蛋白的泌出分析，明确其对效应蛋白泌出的调控及在致病中的功能。预期结果在理论上可揭示P4-ATP酶在疫霉菌致病中的作用机制，在应用上可为作物疫病的防治新策略提供理论依据。

由疫霉菌侵染引起的作物疫病是农业生产上的重要病害，从分子水平上认识疫霉菌的致病机制是疫病控制的基础，其中辣椒疫霉菌是一种具有广泛寄主的重要植物病原疫霉菌。模式生物P4-ATP酶通过调控囊泡形成及蛋白泌出，从而维持正常生命过程，在生长发育中具有重要作用，但是目前关于P4-ATP酶在辣椒疫霉菌中的功能尚未见研究，因此，本研究对辣椒疫霉菌的P4-ATP酶PcAPT1在生长发育及致病中的作用进行研究，结果发现，辣椒疫霉P4-ATP酶基因PcAPT1敲除突变体致病力显著减弱，胞外酶泌出缺陷，表明突变体大大降低了致病性，PcAPT1在致病中具有重要作用；结合不同磷脂种类的磷脂膜染料 (NBD)来有效检测磷脂在膜上的转运能力及转运磷脂的种类，明确PcAPT1介导的磷脂在膜上的转运能力，表明PcAPT1参与了磷脂转运到细胞膜的过程；分析结果表明PcATP1基因与很多基因协同合作共同发挥作用功能；比较野生型和敲除突变体菌株的分泌蛋白组，获得受PcAPT1调控的分泌蛋白，明确PcAPT1对辣椒疫霉菌外泌蛋白质组的影响，表明突变体的水解酶及致病相关蛋白的泌出减少，说明PcAPT1介导了水解酶及致病相关蛋白的外泌，分析同时表明PcAPT1对效应蛋白的外泌具有选择性。最终明确了PcAPT1通过介导效应蛋白的泌出调控辣椒疫霉生长发育及致病性。预期结果在理论上可揭示P4-ATP酶在疫霉菌致病中的作用机制，在应用上可为作物疫病的防治新策略提供理论依据。