葡萄黑痘病菌关键效应蛋白调控葡萄免疫反应的分子机制

葡萄黑痘病是危害欧洲葡萄的重要真菌病害之一，严重影响葡萄的产量和品质。效应蛋白是深入研究葡萄-黑痘菌互作机制的重要桥梁。申请人前期通过葡萄黑痘菌基因组测序、转录组分析、表达分析和瞬时转化实验筛选到8个关键效应蛋白。项目围绕黑痘菌效应蛋白调控葡萄免疫反应机制这一科学问题，拟开展：1）创制葡萄黑痘菌效应蛋白突变菌株，通过形态观察、产孢特性和致病实验筛选1-2个关键效应蛋白；2）利用免疫共沉淀质谱技术和酵母文库在抗病和感病葡萄中筛选互作蛋白，通过酵母回复验证、双分子荧光互补和免疫共沉淀进行互作验证；3）构建靶蛋白超表达载体和基因编辑载体，遗传转化‘无核白’葡萄胚性愈伤组织，获得转基因株系后接种葡萄黑痘病菌，结合病情指数、组织病理学观察、生物量测定和防御相关基因表达解析靶蛋白的抗病功能。研究结果将阐明葡萄黑痘病菌效应蛋白和葡萄靶蛋白协同调控葡萄免疫反应的分子机制，为改善葡萄抗病性提供新路径。

Grape anthracnose is an important fungal disease during grape production for Vitis vinifera. The main impact is on fruit quality and yield. Fungal effectors are an important bridge for in-depth study of Elsinoë ampelina-grape interactions. Eight effectors were selected by genome sequencing, RNA-seq, expression analysis, and transient assays in Nicotiana benthamiana in previous work. The project will focus on the scientific problem of regulating the immune response mechanism of grape by the effector protein of E. ampelina. First, one or two key effector proteins will be obtained from eight effector protein mutants of E. ampelina using morphological observation, sporulation characteristics, and pathogenicity experiments. Second, target proteins of effector protein EaSSPs were screened by co-immunoprecipitation-mass spectrometry and yeast library in the resistant Chinese wild Vitis quinquangularis clone ‘Shang-24’ and susceptible Vitis vinifera ‘Thompson Seedless’, respectively. The candidate interacting proteins were verified by Yeast Two-Hybrid, Bimolecular Fluorescent Complimentary (BiFC), and Co-Immunoprecipitation (CoIP).Target proteins of grape were cloned into the vectors for the over-expression system and CRISPR/Cas9 system, respectively. Transgenic plants will be obtained using embryonic callus of the Thompson seedless cultivar via Agrobacterium-mediated transformation. The biological functions of grape target proteins will be confirmed using inoculation experiments, disease index, histopathological observation, biomass estimation and defense-related gene expression. The final results will reveal the molecular mechanisms of grape immune response mediated by effector protein-grape target protein interactions, and to provide a new way for disease resistance of grape.