辣椒疫霉菌致病必需效应子的多态性与功能分化研究

Phytophthora capsici is an important plant pathogenic oomycete, which can infect hundreds of vegetables and horticultural crops. Pepper blight, caused by P. capsici, is a devastating disease in pepper production due to frequent virulence variation and rapid spread of the pathogen. The disease has become a potential threat to safety production of pepper. Preliminary research in the cooperative laboratory found that PcRxLR207、PcRxLR48、PcCRN4 and PcAvr3a12 are four virulence essential effectors of P. capsici. However, their polymorphisms、variation characteristics and virulence functional differentiation in natural populations are remains unknown. This project aims to analyze the polymorphism of the four effector genes at transcriptional and DNA sequence levels by using RT-PCR combined with gene sequence confirmation on the basis of the previous research on population genetic diversity of P. capsici. Then, the identified candidate variation types of four effector genes will be used to test for its functional differentiation from the following aspects: to test for its effect on inducing plant cell death activity and promoting infection by P. capsici via Agrobacterium tumefaciens-mediated transient expression; to analyze the effects of variation types on H2O2 accumulation as well as expression of pathogenesis-related protein genes in Nicotiana benthamiana; to verify its interaction with known host target proteins and its subcellular localization. Furthermore, the functional differentiation mechanism of effector genes with both polymorphism and functional differentiation are to be revealed. The study is expected to elucidate the mechanism of virulence variation of P. capsici, and to provide theoretical basis for developing a particular way and approach for prevention and control of diseases caused by P. capsici.

辣椒疫霉菌为卵菌，能侵染数百种蔬菜作物。由其引致的辣椒疫病，发病速度快、流行性强、毁灭性大，已成为我国辣椒安全生产的重要威胁。合作实验室前期发现PcRxLR207、PcRxLR48、PcCRN4和PcAvr3a12是辣椒疫霉菌致病关键效应子，但是它们在自然群体中的多态性、变异特征、毒性功能分化以及机制尚未知。本项目拟在前期对该菌群体遗传多样性分析的基础上，采用RT-PCR，结合基因序列测定，分析四个致病关键效应子在转录和序列水平上的多态性；采用根癌农杆菌介导的基因瞬时表达技术，测试不同变异类型在诱导植物细胞死亡活性和促进辣椒疫霉菌侵染中的功能分化；分析其对本氏烟中活性氧累积和病程相关蛋白基因表达的影响；验证其与已知寄主靶标蛋白的互作及亚细胞定位。进一步揭示既有多态性又存在功能分化效应子基因的功能分化机制。研究可望阐明辣椒疫霉菌的毒性变异机制，为开发辣椒疫霉病害防控的新途径提供理论依据。

本研究以田间自然群体中的辣椒疫霉菌为研究对象，从表型和基因型两个层面系统分析了病菌的群体遗传多样性，并从中选取了30个存在遗传差异的辣椒疫霉菌毒性菌系，对4个（PcRxLR207、PcRxLR48、PcCRN4和PcAvr3a12）重要的致病必需效应子基因在转录和序列水平上进行了多态性及其毒性功能分化情况的分析。采用根癌农杆菌介导的基因瞬时表达技术，测试了不同变异类型在诱导植物细胞死亡活性和促进辣椒疫霉菌侵染中的功能分化；分析了其对本氏烟中活性氧累积和病程相关蛋白基因表达的影响；验证了其与已知寄主靶标蛋白的互作及亚细胞定位。选取了1个既有多态性又存在功能分化的效应子基因，进一步进行了功能分化机制的分析。研究结果对阐明辣椒疫霉菌的毒性变异机制，为开发辣椒疫霉病害防控的新途径和新方法提供理论依据。