稻瘟菌可长距离移动的效应蛋白MoSDT1在与水稻互作中的分子鉴定

It is widely accepted as critical to discover mechanism on interaction of plant and pathogen through understanding molecular function of effector proteins during plant infection. Most of identified genes encoding effectors in M.oryzae were of cytoplasmic function and specifically induced defense response on a resistant variety carrying a corresponding resistance gene, while it is few reports that effector protein can carry out long-distance movement within rice plants, and they spontaneously and non-specifically induce defense response on many rice monogenic lines carrying different single resistance gene. On the basis of our previous research, we will construct binary vectors, overexpression vectors and prokaryotic expression vectors of MoSDT1 fusion to mCherry, deletion sequences (such as signal peptide sequence, zinc finger domain sequence and C-terminal sequence fusion to mCherry) of MoSDT1. The transformation strains are got through PEG-mediated method, the pathogenicity test of transformation strains and observing spatial expression and secretion of mCherry fusion protein during plant infection were carried out, which will verify the secreteion character and domain of MoSDT1, it need to assay whether MoSDT1 could enter protoplast of rice through transient expression. Rice roots tips are treated with prokaryotic products to observe translocation of fusion proteins in root tissues, which will demonstrate motif of MoSDT1 required for translocation of proteins, simultaneously, we will analyze whether MoSDT1 localized in apoplast through combined method of plasmolysis and immunocytochemistry. Our study will discover molecular mechanism on function of MoSDT1 during interaction of rice and rice blast fungus.

研究病原菌效应蛋白在病害发展中的功能是揭示病原菌与寄主互作的关键。已鉴定的稻瘟菌效应蛋白大多只诱导持单个或少数几个抗性基因的水稻防御响应，而对能诱导持不同主效抗病基因的多个水稻防御响应且能在水稻植株内长距离移动的效应蛋白的鉴定还未见报道。申请者前期发现一个能诱导持不同抗病基因的单基因系水稻品种防御响应且能在水稻植株内长距离移动的新效应蛋白MoSDT1。在此基础上，拟构建该效应蛋白及其部分序列缺失与mCherry融合的双元表达载体、过表达载体和原核表达载体，分析转化菌株的致病力及观察融合蛋白在稻瘟菌侵染水稻中的行为；通过人工合成多肽处理原生质体，明确与MoSDT1诱导防御响应的功能域；通过观察原核表达产物在水稻植株内的长距离移动，明确与MoSDT1长距离移动相关的必需序列；再结合质壁分离和免疫细胞化学分析该蛋白是在寄主外质体还是细胞质。本研究可揭示MoSDT1在稻瘟菌与水稻互作中的分子机制。

为了成功侵入寄主，病原菌进化或分泌出各种效应蛋白控制寄主代谢或生理过程。研究病原菌效应蛋白在病害发生发展中的功能是有助于揭示病原菌与寄主互作机制。本项目在前期发现一个能诱导持有不同抗病基因的单基因系水稻品种防御响应的稻瘟病菌全新的效应蛋白MoSDT1的基础上，以构建BAS1和BAS4与mCherry融合的原核表达载体和过表达载体为对照，研究了稻瘟病菌效应蛋白MoSDT1与mCherry融合的原核表达产物喷雾处理水稻叶片致使处理的水稻叶片再接种稻瘟病菌发病症状加重；MoSDT1和BAS1在稻瘟病菌株侵染水稻早期分泌进入水稻细胞，是细胞质效应蛋白，BAS4在稻瘟病菌侵染水稻早期分泌进入水稻组织，分布于水稻细胞间隙中，是外质体效应蛋白；初步明确了MoSDT1的信号肽和锌指为功能区，C-端参与了MoSDT1在水稻植株内的转运；MoSDT1、BAS1和BAS4在稻瘟病菌株中过表达增强了菌株的毒性及对外界环境刺激的耐受性。项目明确了效应蛋白在稻瘟病菌毒性及对外界环境刺激耐受性的机制，为今后制定稻瘟病绿色防控策略和方法提供了重要的理论参考。