葡萄霜霉菌效应因子PvCRN91功能分析  

Functional analysis of effector PvCRN91 from Plasmopara viticola

作　　者：孙大运 刘露露 郭泽西 韦淑梅 潘凤英[1] 曲俊杰[1] 尹玲[1] SUN Da-yun;LIU Lu-lu;GUO Ze-xi;WEI Shu-mei;PAN Feng-ying;QU Jun-jie;YIN Ling(Key Laboratory of Guangxi Crop Genetic Improvement and Biotechnology,Guangxi Academy of Agricultural Sciences,Nanning,Guangxi 530007,China)

机构地区：[1]广西农业科学院广西作物遗传改良生物技术重点开放实验室,广西南宁530007

出　　处：《南方农业学报》2025年第2期533-543,共11页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32260662);广西科技基地和人才专项(桂科AD21220116);广西农业科学院基本科研业务专项(桂科2021YT121)。

摘　　要：【目的】克隆葡萄霜霉菌PvCRN91基因并研究其功能,为揭示葡萄霜霉菌的致病机制提供理论参考。【方法】利用分子克隆技术扩增PvCRN91基因全长,并分析其编码蛋白的生物信息学特征。通过本氏烟上瞬时表达效应蛋白PvCRN91,观察其在烟草叶片上的表现和对马铃薯疫霉激发子INF1和小鼠促凋亡蛋白BAX触发细胞坏死的影响。构建pBWA(V)HS-PvCRN91-GFP融合表达载体,在葡萄叶片和烟草叶片瞬时表达,并分别接种葡萄霜霉菌和烟草疫霉,分析其促进病原菌侵染的能力。构建pBI221-PvCRN91-GFP表达载体,并在烟草原生质体瞬时表达,分析PvCRN91蛋白的亚细胞定位情况。构建pBWA(V)HS-PvCRN91-3×Flag融合表达载体,通过免疫沉淀—质谱联用技术(IP-MS)方法筛选与PvCRN91互作的靶蛋白。【结果】PvCRN91基因编码区(CDS)全长351 bp,编码116个氨基酸残基,蛋白相对分子量约13.03 kD,不含有信号肽、核定位序列(NLS)和跨膜结构域,含有1个Crinkler结构域和consen‐sus disorder prediction区域;PvCRN91的二级结构含有α-螺旋(31.90%)、延伸链(22.41%)和无规则卷曲(45.69%)。序列比对发现,在多种疫霉菌、水霉菌、霜霉菌和丝囊霉菌中均存在与PvCRN91蛋白一致性较高(56.60%~74.77%)的序列,推测PvCRN91是一个保守的效应因子。PvCRN91蛋白不具有触发烟草细胞坏死的能力,但能完全抑制INF1和BAX诱导的细胞程序性死亡(PCD)反应。PvCRN91效应因子可促进葡萄霜霉菌和烟草疫霉菌的侵染。PvCRN91蛋白亚细胞定位于细胞质和细胞核,推测其在细胞质或细胞核中与寄主靶蛋白互作,抑制寄主的免疫反应。IP-MS方法初步筛选到252个可能与PvCRN91互作的靶蛋白,通过多个代谢途径影响烟草的生理生化和防御反应。【结论】PvCRN91是一个毒力因子,可能在细胞质或细胞核中操纵寄主的靶蛋白,降低植物的防卫反应,促进病原菌的侵染。【Objective】To clone PvCRN91 gene from Plasmopara viticola and verify its functions,which could provide theoretical reference for revealing the pathogenic mechanism of P.viticola.【Method】The full-length of PvCRN91 gene was amplified using molecular cloning techniques,and the bioinformatics characteristics of encoded protein were analyzed.The effector protein PvCRN91 was transiently expressed on Nicotiana benthamiana.The performance of effector protein PVCRN91 on tobacco leaves and effects on cell necrosis induced by Phytophthora infesfans elicitor and mouse pro-apoptotic protein BAX were observed.The pBWA(V)HS-PvCRN91-GFP fusion expression vector was constructed and transiently expressed in grape leaves and tobacco leaves.The leaves were inoculated with P.viticola and Phytophthora nicotianae to analyze its ability to promote pathogen infection.The pBI221-PvCRN91-GFP expression vector was constructed and transiently expressed in tobacco leaves,and the subcellular localization of PvCRN91 protein was analyzed.The pBWA(V)HS-PvCRN91-3×Flag fusion expression vector was constructed,and the target proteins interacting with PvCRN91 were screened by immunoprecipitation-mass spectrometry(IP-MS).【Result】The coding region(CDS)of gene PvCRN91 was 351 bp,encoding 116 amino acids residues with about 13.03 kD molecular weight.It did not contain signal peptides,nuclear localization sequences(NLS)and transmembrane domains,but contained 1 Crinkler domain and consensus disorder prediction domain.The secondary structure of PvCRN91 consisted ofα-helix(31.90%),extended chain(22.41%)and random coil(45.69%).Sequence comparison showed that there were sequences with high consistency(56.60%-74.77%)with PvCRN91 protein in various phytophthora,water mold,peronospora and aphanomyces,suggesting that PvCRN91 was a conserved effector.PvCRN91 did not have the ability to trigger cell necrosis in tobacco,but completely inhibited INF1-and BAX-induced programmed cell death(PCD)reaction.PvCRN91 effector could promote the infection of P.viti

关 键 词：葡萄霜霉菌 PvCRN91基因 效应因子 Crinkler结构域 防卫反应 

分 类 号：S432.1[农业科学—植物病理学]