抗橡胶树白粉病基因ROH1作用机理的研究

By using the model of Arabidopsis-Oidium heveae interaction, we found that Oidium heveae can trigger hypersensitive response(HR) in Arabidopsis thaliana in an AtEDS1 dependent manner, suggesting that Arabidopsis activate HR though a resistance (R) protein of TIR-NB-LRR (Toll-Interleukin1 Receptor-nucleotide binding-leucine-rich repeat) family upon perception of Oidium heveae. Because transgenic expression of HbEDS1, a closed homolog of AtEDS1 in Hevea brasiliensis, can functionally complement the decreased resistance to Oidium heveae caused by AtEDS1 mutation in Arabidopsis, implying that the resistance mechanisms in Arabidopsis are probably suitable for Hevea brasiliensis. Recently, a ROH1 (Resistance to Oidium heveae 1) gene was isolated by screening Arabidopsis T-DNA insertional mutants of 58 TIR-NB-LRR genes. Loss-of-function mutation of ROH1 remarkably delayed the resistance to Oidium heveae in Arabidopsis. Furthermore, a ROH1 binding protein, RBP1, was identified by a yeast-two hybrid screening. We found that RBP1 also contributes to Oidium heveae-triggered resistance in Arabidopsis. Thus, in the project, we will further determine the interaction between ROH1 and RBP1 by using the Immunoprecipitation and Split-YFP methods. And, the molecular mechanisms employed by ROH1 and RBP1 to activate resistance against Oidium heveae will be explored through genetics, cytology and biochemistry methods. In a conclusion, this project is to make the functional study of ROH1 gene and lay the fundation for the future prevention of powdery mildew in Hevea brasiliensis.

以拟南芥-橡胶树白粉菌为宿主-病原菌研究模式，申请人前期发现拟南芥能够通过触发依赖于AtEDS1的超敏反应来抵御橡胶树白粉菌的侵染，表明拟南芥可能通过TIR-NB-LRR类抗性（R）蛋白启动对橡胶树白粉菌的抗性。由于AtEDS1在橡胶树上的同源基因HbEDS1可以功能互补拟南芥AtEDS1突变引起的抗性减弱，我们推测橡胶树存在与拟南芥类似的抗性机制。前期，申请人通过对拟南芥58个编码TIR-NB-LRR结构蛋白的R基因突变体进行筛选，发现ROH1基因突变引起拟南芥对橡胶树白粉菌抗性显著降低。通过酵母双杂交筛选，我们获得了ROH1互作蛋白RBP1。本项目将利用免疫共沉淀和双分子荧光互补等方法进一步分析ROH1与RBP1的互作，并通过遗传学、细胞学和生物化学等方法深入研究二者参与拟南芥对橡胶树白粉菌抗性的分子机制。该研究为将来解决白粉菌引起的橡胶树病害提供重要理论基础和潜在的应用价值。

橡胶树白粉菌在拟南芥上激发抗病反应，并且该抗病反应依赖于EDS1和PAD4，推测TIR-NB-LRR类抗病基因参与拟南芥对橡胶树白粉菌抗病性。本课题通过对58个拟南芥TIR-NB-LRR类突变体纯合系进行筛选，发现ROH1基因参与拟南芥对橡胶树白粉菌的抗病性，并从遗传学角度证实了该基因的抗病功能。进一步研究发现ROH1基因对烟草白粉病菌UMSG3和番茄白粉病菌UCSC1具有广谱抗病性，但不参与拟南芥对假单胞菌效应蛋白激发的抗病过程。另外，通过生物化学，细胞生物学和遗传学技术，发现ROH1基因主要定位于植物细胞膜，受橡胶树白粉菌的诱导下调表达，过量表达ROH1基因可以在植物内激发大量的细胞死亡，并且ROH1与EDS1和RBP1蛋白在植物体内发生两两相互作用。综合上述结果，本研究为进一步解释TIR-NB-LRR类基因抗白粉病信号通路和实现橡胶树白粉病抗病遗传育种提供了重要的理论依据。项目资助发表SCI论文3篇，中文核心期刊4篇，授权国家发明专利1项，申请国家发明专利1项，待发表SCI论文1篇。培养青年教师2名晋升副教授，硕士研究生3名，1名已取得硕士研究生学位。该项目共获得批准经费41万元，已支出经费31.70万元，各项支出基本与预算相符。剩余经费9.3万元，剩余经费将用于本项目研究后续支出。