小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应

doi:10.3864/j.issn.0578-1752.2020.12.006

摘要/Abstract

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（Type Ⅲ secretion 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胞质,表明其作用于细胞内。Pt18906在单基因系抗病品种TcLr27+31和TcLr42上能够引起过敏性坏死反应,表明该效应蛋白的无毒性,Pt18906能够引起TcLr27+31中胼胝质的积累和活性氧的迸发,胼胝质随注射时间的增加而逐渐积累,活性氧在注射后的10 min达到最高。【结论】位于28—47位的氨基酸决定Pt18906的毒性主要功能,Pt18906能激发小麦TcLr27+31双层防御反应。

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

Abstract:

【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 in the cDNA of wheat leaves inoculated by Pt strain 13-5-72. The effector protein lacks known functional domains and motifs. Its working environment is alkaline. Pt18906 could inhibit the PCD induced by Bax from mice and INF1 from Oomycetes by transient expression in N. benthamiana cells, which indicates that the effector protein has virulent function. By constructing deletion mutants, it is clear that the 28-47 amino acids have an important role in its virulent function. The effector protein is located in the nucleus and cytoplasm, which indicates that it acts within the cells. Pt18906 could cause allergic necrosis in the disease-resistant varieties of single gene lines TcLr27+31 and TcLr42, indicating that the effector protein is avirulent. Pt18906 could cause the accumulation of reactive oxygen species (ROS) and callose in TcLr27+31 through transiently expressing using a bacterial type III secretion system. The callose increased gradually with the injection time, and ROS reached the highest level at 10 min after injection. 【Conclusion】 The main functional domain of Pt18906 is 28-47 amino acids. Pt18906 can trigger the two-layer defense reaction accompanied with the accumulation of callose in wheat TcLr27+31 and oxidative burst.

Key words: Puccinia triticina (Pt), wheat leaf rust, effector protein, callose, reactive oxygen species (ROS), pathogenicity, defense reaction

齐悦,吕峻元,张悦,韦杰,张娜,杨文香,刘大群. 小麦叶锈菌效应蛋白Pt18906激发TcLr27+31的双层防御反应[J]. 中国农业科学, 2020, 53(12): 2371-2384.

QI Yue,LÜ JunYuan,ZHANG Yue,WEI Jie,ZHANG Na,YANG WenXiang,LIU DaQun. Puccinia triticina Effector Protein Pt18906 Triggered Two-Layer Defense Reaction in TcLr27+31[J]. Scientia Agricultura Sinica, 2020, 53(12): 2371-2384.

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