南方农业学报2025，Vol.56Issue(2)：533-543,11.DOI:10.3969/j.issn.2095-1191.2025.02.017
葡萄霜霉菌效应因子PvCRN91功能分析
Functional analysis of effector PvCRN91 from Plasmopara viticola
摘要
Abstract
[Objective]To clone PvCRN91 gene from Plasmopara viticola and verify its functions,which could pro-vide 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 effec-tor 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 con-structed 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 of α-helix(31.90%),extended chain(22.41%)and random coil(45.69%).Sequence comparison showed that there were sequences with high consis-tency(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 pro-mote the infection of P.viticola and P.nicotianae.PvCRN91 protein subcellular location was in cytoplasm and nucleus,and it was speculated that it interacted with host target protein in cytoplasm or nucleus to inhibit host immune response.IP-MS method initially screened 252 target proteins that might interact with PvCRN91 and affect the physiological,bio-chemical and defense responses of tobacco through multiple metabolic pathways.[Conclusion]PvCRN91 is a virulence factor that may manipulate host target proteins in the cytoplasm or nucleus to reduce plant defense responses and promote pathogen infection.关键词
葡萄霜霉菌/PvCRN91基因/效应因子/Crinkler结构域/防卫反应Key words
Plasmopara viticola/PvCRN91 gene/effect factor/Crinkler domain/defense response分类
农业科技引用本文复制引用
孙大运,刘露露,郭泽西,韦淑梅,潘凤英,曲俊杰,尹玲..葡萄霜霉菌效应因子PvCRN91功能分析[J].南方农业学报,2025,56(2):533-543,11.基金项目
国家自然科学基金项目(32260662) （32260662）
广西科技基地和人才专项(桂科AD21220116) （桂科AD21220116）
广西农业科学院基本科研业务专项(桂科2021YT121) National Natural Science Foundation of China(32260662) （桂科2021YT121）
Guangxi Science and Technology Base and Talent Special Project(Guike AD21220116) （Guike AD21220116）
Basic Research Project of Guangxi Academy of Agricultural Sciences(Guinongke 2021YT121) （Guinongke 2021YT121）
