簇毛麦CMPG1-V与ToxABP1s蛋白互作共同调控小麦白粉病抗性的分子机制解析

Powdery mildew (Pm) is the second fungal disease endangering wheat production in China. The resistance of wheat cultivars could be rapidly overcome by frequent emergence of new virulence isolates of the pathogen Blumeria graminis f. sp tritici. The identification of broad-spectrum resistance (BSR) genes and the elucidation of molecular mechanism underlying BSR is very important for improvement of Pm resistance. Wheat relative Haynaldia villosa shows BSR to Pm. Thus, provide an ideal model system for the molecular mechanism study of BSR. In previous study, an E3 ligase gene CMPG1-V was isolated from H. villosa, and was proved to positively regulate broad-spectrum resistance to Pm. To elucidate the mechanism mediated by CMPG1-V, a yeast two-hybrid library was screened and a chloroplast localized protein ToxABP1-V was identified to be interacted with CMPG1-V. Prelimary results from gene expression and transient gene silencing assay indicated ToxABP1s may negatively regulate Pm resistance. In the proposed research, its function in Pm resistance will be further characterized by VIGS and genome editing, and light signal regulation on Pm resistance will be studied. The potential values of the ToxABP1 genes in improvement for Pm resistance will be evaluated. The mechanism of ToxABP1s and CMPG1-V interaction and the relationship of the interaction with Pm resistance will be clarified by point mutation and ubiquitination assay, etc. The implementation of the work will not only provide new gene resource for wheat improvement for disease resistance, but also facilitate our understanding of plant innate immunity.

白粉病是我国小麦第二大真菌病害。由于白粉菌生理小种变异频繁，品种抗性丧失快，因此广谱、持久抗性基因的发掘利用及抗性机制解析意义重大。小麦近缘物种簇毛麦广谱高抗白粉病，解析其抗性机制可为小麦广谱抗性改良提供依据。前期研究发现，来自簇毛麦的E3连接酶CMPG1-V正向调控小麦白粉病广谱抗性。通过酵母双杂交筛选，发现叶绿体定位蛋白ToxABP1-V与CMPG1-V互作，基因表达、TIGS初步表明，ToxABP1s基因负向调控小麦白粉病抗性。本项目拟在此基础上，利用VIGS、基因编辑等技术研究ToxABP1s基因在抗白粉病中的功能，解析光信号调控与抗病通路的关系；明确其在抗白粉病基因工程中的利用价值；利用点突变、泛素化等实验解析ToxABP1s与CMPG1-V的互作机制，以及该互作在小麦白粉病抗性中的作用。本研究成果，既可为植物抗病育种提供基因资源，也将丰富植物先天免疫的分子机制理论。

本项目紧紧围绕三个研究内容，分析了ToxABP1s基因的表达模式；利用TIGS、VIGS、CRISPR-CAS9、转基因等技术阐明了ToxABP1类基因在白粉病抗性中的功能；利用点突变、泛素化实验等技术解析ToxABP1s与CMPG1-V的互作机制，其互作在小麦白粉病抗性中的功能；解析了光信号调控ToxABP1s介导抗病通路。本项目全面完成了预定的研究内容，达到了预期目标。取得的主要进展如下：（1）在簇毛麦以及小麦中克隆ToxABP1s类基因，发现ToxABP1s基因受白粉病诱导下调表达；通过TIGS、VIGS、CRISPR-CAS9、转基因等技术明确了ToxABP1s基因负向调控小麦白粉病抗性，其中植物激素SA和信号分子H2O2参与ToxABP1s基因介导的白粉病抗性。（2）发现ToxABP1s基因存在可变启动子（alternative promotor），转录、翻译产生两种蛋白（定位于叶绿体的ToxABP1-VFL蛋白和定位于细胞质和细胞核的ToxABP1-VCyt蛋白）。酵母双杂交、co-IP和Bifc结果发现：CMPG1-V特异与ToxABP1-VCyt蛋白互作，不与ToxABP1-VFL蛋白互作；ToxABP1-V通过其CC domain与CMPG1-V的ARM domain互作。体外泛素化实验、体外蛋白降解实验表明：CMPG1-V通过多聚泛素化ToxABP1-V，促进其被26S蛋白酶体降解。（3）tatoxabp1突变体光合作用效率显著降低。tatoxabp1突变体自发积累ROS和细胞坏死，其部分依赖于光照。申请并获批国家发明专利1件，培养毕业研究生4名。