3个TIR-NB-LRR类抗橡胶树白粉病基因协同作用机理的研究

It has been reported that Arabidopsis accession Lag2-2 showed significant susceptibility to Oidium heveae (O. heveae), and recently we found there is a stop mutation of the Toll-Interleukin1 Receptor (TIR)-nucleotide binding (NB)-leucine-rich repeat (LRR) protein named ROH1（Resistance to O. heveae）in Lag2-2 by the genomic sequencing. Further study confirmed that ROH1 contributes to the disease resistance to O. heveae. Through the genome localization analysis of ROH1, another two TIR-NB-LRR-type genes ROH2 and ROH3 reside within a single locus with ROH1. Loss-of-function mutations of ROH2 and ROH3 also displayed remarkable susceptibility to O. heveae. In addition, Co-immunoprecipitation assay showed that ROH1 can interact with ROH3 in Arabidopsis protoplast, indicating that ROH1, ROH2 and ROH3 may function together in disease resistance against O. heveae. Thus, in this project, we will further determine the interactions between ROH1, ROH2 and ROH3 by Co-immunoprecipitation and pull down assays and confirm the interaction domains of these three proteins. Also, the co-function mechanisms employed by ROH1,ROH2 and ROH3 in resistance against O. heveae will be explored through genetics, cytology methods. Overall, this study will provide a basis for future prevention of powdery mildew caused by O. heveae in rubber trees.

橡胶树白粉菌在拟南芥生态型Lag2-2表现抗性减弱的表型，全基因组测序发现Lag2-2 TIR-NB-LRR类抗性蛋白ROH1发生了终止突变，表型分析进一步证实ROH1参与拟南芥对橡胶树白粉菌的抗病性。通过对ROH1所在基因组上下游序列分析，发现另外两个TIR-NB-LRR类基因ROH2和ROH3与ROH1位于同一基因簇内，同时，roh2和roh3突变体也表现对橡胶树白粉菌感病性提高的表型，免疫共沉淀试验证实ROH1和ROH3发生相互作用，推测ROH1，ROH2和ROH3通过协同作用实现拟南芥对白粉菌的抗病性。本项目在此工作的基础上，进一步通过免疫共沉淀和pull down试验确定ROH1，ROH2和ROH3 3个蛋白之间是否存在两两互作及互作结构域，并从遗传学和细胞学角度深入研究3个蛋白的抗性机理和潜在的协同作用机制，为防治橡胶树白粉病奠定坚实的理论基础。

生态型Lag2-2全基因组测序和表型分析，发现TIR-NB-LRR类抗性蛋白ROH1参与了拟南芥对橡胶树白粉菌的抗病性。对ROH1所在基因组上下游序列分析，找到另外两个TIR-NB-LRR类基因ROH2和 ROH3与ROH1位于同一基因簇内，本课题通过在roh1，roh2和roh3单突变体，以及roh1/roh2/roh3 3突变体接种橡胶树白粉菌和番茄白粉病菌，发现3个基因不同程度地参与了拟南芥对白粉菌的抗病性，并且存在功能冗余。进一步从遗传学角度证实了3个基因的抗病功能。另外，通过生物化学，细胞生物学技术，发现ROH1基因主要定位于植物细胞膜，ROH2和ROH3定位于细胞膜和细胞质，3个基因受橡胶树白粉菌的诱导下调表达。利用免疫共沉淀，ROH1与ROH3在植物体内相互作用，但ROH1与ROH2以及ROH2与ROH3在植物体内不发生相互作用，这为进一步探索3个基因的协同作用机制奠定了基础。项目资助发表SCI 论文2篇，中文核心期刊2篇，授权国家发明专利1项，申请国家发明专利1项，投稿论文1篇。培养硕士研究生5名，3名已取得硕士研究生学位。项目投入经费36万元，支出经费22.84万元，各项支出基本与预算相符。剩余经费13.16万元，剩余经费将用于本项目研究后续支出。