葡萄霜霉菌RxLR31154效应因子与中国野生葡萄OEE2蛋白互作生物学功能研究

Downy mildew, caused by the Oomycete Plasmopara viticola, is one of serious fungal diseases in the cultivated European grapevines (Vitis vinifera L.). Pathogen effectors are known as key factor when they are recognized by plants. Effectors of the Oomycete P. viticola play an important role in the process of pathogenesis. At present study, an effector of RxLR31154 was isolated from P. viticola, and is able to inhibit the programmed cell death caused by induced by mouse pro-apoptosis protein BAX in N. benthamiana, identifying that the RxLR31154 of P. viticola effectors maybe play an important role in pathogehicity. In the following study, a cDNA library, constructed using diseased leaf tissues of Vitis piasezkii ‘Liuba-8’ infected with P. viticola, were screened for proteins interacting with the target effector protein using a yeast two-hybrid approach. A candidate protein of OEE2 was obtained by screening cDNA library clones and was confirmed by co-transformation to be effector-interacting in vitro and in vivo using yeast two-hybrid and Co-immunoprecipitation approaches. To understand the effection of RxLR31154 on subcellular localization of OEE2 protein, the quantity of OEE2 protein in chloroplast and photosynthesis in host plant, we generated transgenic N. benthamina plants overexpressing OEE2 (35S-OEE2) and transgenic N. benthamiana plants carrying a hairpin-structure OEE2 expression cassette (OEE2-RNAi). The structure of chloroplast and photosynthesis were analyzed in RNAi N. benthamina plants, and to investigate on the biological role of interaction between RxLR31154 and OEE2. OEE2-silenced N. benthamiana plants，35S-OEE2 N. benthamiana plants and WT plants were then mechanically inoculated with genetic modification of Phytophthora capsici L. The development of symptom appearance will be evaluated as compared with WT, meanwhile, the pathogenicity of RxLR31154 gene was further analyzed in the transferred European grapevine plants inoculated by P. viticola. The goals are to identify the pathogenicity of effectors from the Oomycete P. viticola and the mechanism of defense response in host plant at the molecular level. This study facilitates further investigation on translocation of effector and its virulence mechanisms in host plant cells and will facilitate the development of novel disease control strategies.

葡萄霜霉病是危害最严重的葡萄真菌病害之一，霜霉菌效应因子在抑制葡萄防御反应中起关键作用。本研究是在鉴定RxLR31154效应因子抑制BAX诱导的烟草叶片细胞过敏性死亡和通过酵母双杂交和蛋白质免疫共沉淀验证了RxLR31154与葡萄OEE2蛋白在体外和体内互作的基础之上。分析RXRL31154效应因子对寄主OEE2蛋白叶绿体定位的影响，利用转基因和RNAi技术获得OEE2过表达和表达下调的本氏烟草植株，分析OEE2表达下调的植株叶片中叶绿体结构和光合作用的变化。过表达和表达下调的OEE2转基因烟草植株进行接种转RxLR31154效应因子基因的辣椒疫霉菌侵染效率测定，分析RXRL31154与OEE2互作的生物学功能；同时将OEE2基因过表达和RNAi干扰载体转化欧洲葡萄‘无核白’获得转基因植株，接种霜霉菌，揭示葡萄霜霉菌效应因子致病性和寄主的防卫反应机理。

葡萄霜霉病是由葡萄霜霉菌引起的，严重影响葡萄的品质和产量。我国是葡萄属植物最重要的起源地之一，拥有丰富的野生葡萄种质资源。因此，研究中国野生葡萄种质的抗病性，对葡萄抗病育种具有重要的意义。病原菌通过分泌效应因子改变寄主植物细胞进程和免疫反应，来达到侵染寄主植物的目的，因此研究葡萄霜霉菌效应因子致病机理，探究葡萄-霜霉菌互作的分子机制对抗病育种具有重要的意义。. 葡萄霜霉菌RXLR31154效应因子通过稳定葡萄叶片中VpOEE2蛋白，激活葡萄体内单线态氧信号通路，同时抑制病原菌侵染葡萄叶片诱导的H2O2积累，促进霜霉菌在葡萄叶片中的定殖。本研究鉴定了类效应因子RXLR31154在霜霉菌侵染‘黑比诺’和‘留坝-8’前期均上调表达。过表达RXLR31154转基因葡萄能够促进霜霉菌在葡萄中的侵染。以RXLR31154为诱饵，从复叶葡萄‘留坝-8’叶片接种霜霉菌后的酵母cDNA文库中筛选到一个互作蛋白VpOEE2。VpOEE2由核基因PsbP1编码，是光合系统Ⅱ放氧复合物的主要组成成分。通过酵母双杂交、双分子荧光互补、GST pull down和免疫共沉淀实验验证RXLR31154和VpOEE2互作。蛋白免疫印迹实验表明：OEE2蛋白在‘黑比诺’和‘留坝-8’接种霜霉菌中后期诱导表达。遗传分析实验表明：过表达VpOEE2转基因葡萄和烟草株系分别增强了葡萄和烟草对葡萄霜霉菌和辣椒疫霉菌的感病性，烟草NbPsbP干扰株系能够增强烟草对辣椒疫霉菌的抗性。亚细胞定位及叶绿体分离实验表明：RXLR31154与VpOEE2共定位于叶绿体。叶绿素荧光测定结果表明：与野生型相比，过表达RXLR31154和VpOEE2转基因株系的叶绿素荧光参数Fv/Fm、Y(Ⅱ)、NPQ和ETR没有明显差异，而烟草NbPsbP干扰系的叶绿素荧光参数明显降低。体内蛋白质降解实验表明：RXLR31154能够稳定VpOEE2蛋白。qRT-PCR实验结果表明：接种霜霉菌后过表达RXLR31154和VpOEE2转基因葡萄株系中H2O2清除系统相关基因表达高于野生型，单线态氧1O2信号通路相关基因EX1、EX2和SIB2在转基因株系中的表达量明显高于野生型。DAB染色结果表明：接种霜霉后120 hpi，转基因株系中H2O2积累低于野生型。