小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应  

Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31

作　　者：齐悦 吕峻元 张悦[1] 韦杰 张娜[1] 杨文香[1] 刘大群[2] QI Yue;Lü JunYuan;ZHANG Yue;WEI Jie;ZHANG Na;YANG WenXiang;LIU DaQun(Wheat Leaf Rust Research Center,College of Plant Protection,Agricultural University of Hebei/Technological Innovation Center for Biological Control of Plant Diseases and Insect Pests of Hebei Province/National Engineering Research Center for Agriculture in Northern Mountainous Areas,Baoding 071001,Hebei;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[1]河北农业大学植物保护学院小麦叶锈病研究中心/河北省农作物病虫害生物防治技术创新中心/国家北方山区农业工程技术研究中心,河北保定071001 [2]中国农业科学院研究生院,北京100081

出　　处：《中国农业科学》2020年第12期2371-2384,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(301571956,301871915);国家重点研发计划(2017YFD70700);河北省产业体系小麦创新团队(2018010204)。

摘　　要：【目的】由小麦叶锈菌(Puccinia triticina)引起的小麦叶锈病是影响小麦生产的主要病害之一,在小麦与叶锈菌互作的过程中病菌向寄主细胞分泌效应蛋白,以调控寄主防御反应、发挥毒性功能。开展对小麦叶锈菌效应蛋白的研究,探索小麦叶锈菌的致病机制,为病害的持续防控提供依据。【方法】以小麦叶锈菌13-5-72与感病品种Thatcher互作的cDNA为模板扩增效应蛋白Pt18906,通过SignalP 4.1、TargetP 1.1、TMHMM 2.0和EffectorP 2.0软件对Pt18906进行序列特征分析,利用在线软件Swiss-Model预测Pt18906的三级结构,利用在线软件SOPMA预测Pt18906的二级结构。采用实时荧光定量PCR对Pt18906的表达模式进行分析,借助于烟草的异源表达系统对Pt18906进行抑制Bax和INF1诱导的细胞程序性死亡(programmed cell death,PCD)能力验证,利用酵母系统验证Pt18906的信号肽是否具有分泌功能,采用氨基酸逐步缺失的方法缺失突变Pt18906,从而确定其功能毒性motif;通过在烟草中瞬时表达Pt18906-GFP融合蛋白,结合质壁分离技术分析Pt18906的亚细胞定位,得出效应蛋白的作用位点;利用瞬时表达技术在以Thatcher为背景的不含抗病基因和含有不同抗病基因的全套近等基因系上开展Pt18906无毒性功能分析;采用细菌三型分泌系统(TypeⅢsecretion system)介导的瞬时转化分析Pt18906对寄主防御反应的调控。【结果】从小麦叶锈菌13-5-72与感病品种Thatcher互作6 d的转录组文库中获得一个在接种24 h后显著高表达的、基因全长序列672 bp、编码223个氨基酸的候选效应蛋白Pt18906,该效应蛋白缺乏已知的功能结构域和保守基序,工作环境偏碱性,在烟草细胞中瞬时表达Pt18906,Pt18906能够抑制Bax和INF1诱导的细胞程序性死亡,表明该效应蛋白具有毒性功能,并且通过构建缺失突变体明确其28—47位氨基酸对其毒性功能具有重要作用,该效应蛋白定位于细胞核和细�【Objective】Wheat leaf rust caused by Puccinia triticina(Pt)is one of the main diseases affecting wheat production.In the process of interaction between wheat and Pt,the Pt will secrete some effector proteins to the host cells to regulate the host and play a virulent function.The objective of this study is to carry out the research on Pt effector proteins,explore the pathogenic mechanism of Pt,and to provide a basis for the continuous prevention and control of diseases.【Method】The cDNA of Pt 13-5-72 interacting with susceptible variety Thatcher was used as template to amplify effector protein Pt18906.Sequence features of Pt18906 were analyzed using SignalP 4.1,TargetP 1.1,TMHMM 2.0 and EffectorP 2.0 software.The online software Swiss-Model was used to predict the tertiary structure of Pt18906.The secondary structure of Pt18906 was predicted by online software SOPMA.The expression pattern of Pt18906 was analyzed by RT-qPCR.The verification that Pt18906 inhibited Bax and INF1-induced programmed cell death(PCD)was conducted using Nicotiana benthamiana heterogeneous expression system.The secretion function of Pt18906 signal peptide was verified by the yeast system.The deletion mutation of Pt18906 was used to identify the virulence function motif.Subcellular localization of Pt18906 was analyzed using transient expression of Pt18906-GFP in N.benthamiana cells.Transient expression technology was used to carry out the avirulent function of Pt18906 on a full set of near-isogenic lines that did not contain disease resistance genes and contained different disease resistance genes with Thatcher as the background.The transient transformation of wheat mediated by the TTSS(Type Ⅲ secretion system)was used to analyze the activity of regulating the host defense response of Pt18906【.Result】A Pt effector Pt18906,significantly upregulated during the interaction between Pt and Thatcher,was obtained from the transcriptome library of interaction between Pt and Thatcher.The full-length ORF sequence with 672 bp was amplified

关 键 词：小麦叶锈菌 小麦叶锈病 效应蛋白 胼胝质 活性氧 致病性 防御反应 

分 类 号：S435.121.43[农业科学—农业昆虫与害虫防治]