Xpm Ⅲ型效应蛋白XopAG的生物信息学分析及功能验证  

Bioinformatics Analysis and Functional Validation of Type Ⅲ Effector Protein XopAG of Xpm

作　　者：向黎 朱寿松 耿越 贾素行 张肖飞 李春霞[1,2] 陈银华[1,2] Xiang Li;Zhu Shousong;Geng Yue;Jia Suhang;Zhang Xiaofei;Li Chunxia;Chen Yinhua(Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresource,Hainan University,Haikou,570228;School of Tropical Crops,Hainan University,Haikou,570228;International Center for Tropical Agriculture,Cali,760046,Colombia)

机构地区：[1]海南大学,海南省热带生物资源可持续利用重点实验室-省部共建国家重点实验室培育基地,海口570228 [2]海南大学热带作物学院,海口570228 [3]国际热带农业中心,哥伦比亚卡利760046

出　　处：《分子植物育种》2023年第11期3579-3588,共10页Molecular Plant Breeding

基　　金：国家重点研发项目(2019YFD1001105,2018YFD1000500);国家木薯产业体系项目(CARS-11-HNCYH);海南省研究生创新科研课题(Qhys2021-165)共同资助。

摘　　要：木薯细菌性枯萎病(cassava bacterial blight, CBB)是由菜豆黄单胞菌木薯萎蔫致病变种(Xanthomonas phaseoli pv. manihotis, Xpm)引起的细菌性病害。XopAG是Xpm分泌的一类Ⅲ型效应蛋白,其生化功能和致病分子机制尚不明确。XopAG基因全长1 305 bp,编码434个氨基酸。经在线网站预测,XopAG蛋白有48个磷酸化位点,没有信号肽,不存在跨膜结构域,蛋白二级结构由43.32%的α-螺旋、9.91%的延伸链、5.07%的β-折叠和41.71%的无规则卷曲组成。同源序列比对显示,不同细菌属的XopAG蛋白同源性较低,黄单胞菌属中不同致病变种XopAG蛋白相似性较高。保守结构域分析表明,不同病原菌的XopAG蛋白序列中均含有高度保守的cyclophilin-binding基序(GPxL),该基序能结合亲免蛋白CYP,使寄主植物产生抗性。进化树分析表明,黄单胞菌中不同致病变种的XopAG蛋白亲缘关系相对比较接近,Xpm CHN11编码的XopAG与褐色黄单胞菌莱檬亚种(Xanthomonas fuscans ssp. aurantifolii group C, Xfa-C)亲缘关系最近。采用农杆菌沾花法获得转XopAG基因拟南芥,接种丁香假单胞杆菌番茄致病变种(Pseudomonas syringae pv. tomato str. DC3000, Pst DC3000),发现转XopAG基因拟南芥对Pst DC3000的感病性增强,说明效应蛋白XopAG具有毒性功能。本研究为进一步研究Ⅲ型效应蛋白XopAG的致病机理奠定了基础。Cassava bacterial blight(CBB)is a bacterial disease caused by Xanthomonas phaseoli pv.manihotis(Xpm).The biochemical function and pathgenic mechanism of XopAG,a type II effector protein secreted by Xpm,remain unclear.The XopAG gene is 1305 bp in length and encodes 434 amino acids.According to the prediction of online website,XopAG protein has 48 phosphorylation sites,and there is no signal peptide sequence and trans-membrane domain.The secondary structure of XopAG protein contains 43.32%alpha helix,9.91%extended strand,5.07%beta turn and 41.71%random coil.Homologous sequence comparison showed that XopAG proteins of different bacterial genera had low homology whereas XopAG proteins of different pathogenic variants in Xanthomonas genus had high similarity.Conserved structural domain analysis showed that the XopAG protein sequences of different pathogenic bacteria contain a highly conserved cyclophilin-binding motif(GPxL),which binds cyclophilin and induces the host plant resistance.Evolutionary tree analysis demonstrated that the affinities of different pathogenic variants in Xanthomonas are relatively close.The Xpm CHN11-encoded XopAG is most closely related to Xanthomonas fuscans ssp.aurantifoli group C(Xfa-C).The XopAG-transgenic Arabidopsis thaliana was obtained by the Agrobacterium tumefaciens flower-dip method and inoculated with Pseudomonas syringae pv.tomato str.DC3000(Pst DC3000).The results showed that compare to WT Arabidopsis thaliana,XopAG-transgenic Arabidopsis thaliana had a higher susceptibility to Pst DC3000,indicating that the effector protein XopAG has virulence function.This study lays the foundation for further research on the pathogenic mechanism of the type Ⅲ effector protein XopAG.

关 键 词：菜豆黄单胞菌木薯萎蔫致病变种 XopAG Ⅲ型效应蛋白 转基因拟南芥 

分 类 号：S435.33[农业科学—农业昆虫与害虫防治]