樱桃茎腐病菌RxLR效应分子PpAvh241功能与作用机制研究

In this project, we will study on the function and signaling pathway of RxLR effector PpAvh241 of the pathogen Phytophthora parasitica of cherry stem rot. RxLR effectors played an important role during the interaction between pathogen and host. In this paper, a large number of RxLR candidate effectors were obtained by screening and analyzing in the early stage. Further experiments revealed that a typical RxLR effector candidate, PpAvh241, was localized on the cell membrane of the plants. It is presumed that PpAvh241 has an important toxic function during infection based on the preliminary experimental results and bioinformatics analysis. However, its function and mechanism need to be further explored. We propose using of P. parasitica transient silencing transformation system to gain the PpAvh241 gene silencing mutants and analyze their phenotypes, over expressing the protein on Nicotiana benthamiana to analyze the function and subcellular localization, testing the virulence of P. parasitica silencing mutants to host plants and the expression of genes related to disease resistance in plants, Screening of interacting proteins of the RxLR effector Avh241, finally confirm the function and mechanism of PpAvh241 during the host-pathogen interaction.

本项目拟开展樱桃茎腐病病原菌寄生疫霉RxLR效应分子PpAvh241在植物细胞中功能及作用机制的研究。RxLR效应分子在病原菌与寄主互作过程中有非常重要的作用。申请者前期通过对寄生疫霉基因组进行大量生物信息学筛选和分析，得到了一批候选RxLR效应分子。进一步实验发现其中一个典型的RxLR效应分子PpAvh241定位在植物的细胞膜上，根据初步实验结果和生物信息学分析该效应分子在致病过程中有重要功能。我们拟利用寄生疫霉瞬时沉默系统、烟草-病毒载体瞬时过表达系统、寄生疫霉-寄主植物互作系统和酵母双杂交系统，分别在烟草上过表达该效应蛋白、沉默寄生疫霉PpAvh241基因得到沉默转化子进行表型分析、沉默转化子对烟草、拟南芥及寄主植物樱桃的致病力的变化分析、植物抗病相关基因表达检测及效应蛋白的互作靶蛋白的筛选等实验，最终确定PpAvh241在侵染过程中的功能及作用机制。

樱桃茎腐病能够引起樱桃苗叶片和茎部枯萎至整株死亡。研究樱桃与病原菌的互作机制、田间发生规律及防控技术能够为病害防控手段的提出提供更科学的理论依据。从田间采集樱桃茎腐病病株，经过分离纯化得到了疑似病原菌的纯培养物，经柯赫氏法则、生理生化及分子生物学鉴定，明确了樱桃茎腐病的病原菌为烟草疫霉；测定了不同品种樱桃对烟草疫霉的抗感病情况，进一步对抗病品种和感病品种的樱桃叶片厚度、气孔大小、抗性酶活性进行测定，初步明确了樱桃在抗烟草疫霉过程中的关键因素；通过对侵染前后烟草疫霉转录组数据分析，明确了其外泌效应蛋白（RxLR、NLP、Crinker (CRN)、Elicitin 、Transglutaminase elicitor、Enzyme inhibitor等）在菌丝阶段、游动孢子阶段、侵染前期及侵染后期表达量的变化，并初步筛选出了一批侵染前后表达差异较大的RxLR效应分子；PpAvh241作为烟草疫霉中的一个典型的RXLR效应分子，与大豆疫霉PsAvh241相似度达60%左右。通过对该效应分子功能分析，明确了其在植物中的亚细胞定位及功能；通过在不同MAPK关键基因沉默的烟草叶片中瞬时过量表达PpAVH241蛋白，初步分析了该效应分子在烟草中作用的信号通路；通过田间和室内实验进行调查研究，初步建立了樱桃茎腐病的田间防控技术体系。