细菌性条斑病菌效应蛋白XopE35抑制水稻免疫的分子机理

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola is one of the important diseases in rice. The secreted effectors are essential for full virulence of the pathogen. Therefore, understanding of molecular mechanisms underlying virulence functions of the effectors will facilitate the development of novel and effective strategies for disease management. Previous results showed that the effector XopE35 is universally present in Xanthomonas spp. and suppresses plant immunity. More interestingly, preliminary results showed that the effector interacts not only with JAZ proteins, key regulators of Jasmonate (JA) signaling, and also with SLR1, the key regulator of gibberellin (GA) signaling. In this study, we will attempt to confirm the interactions between XopE35 and its candidate interactors using various approaches, such as immunoprecipitation. It will be also investigated whether the functions and stability of these target proteins are affected by XopE35 under semi-in vitro and in vivo conditions. Transcriptome analysis by RNA-seq on the constructed transgenic rice plants with XopE35 expression will be performed to determine the effect of XopE35 on the crosstalk between JA and GA signaling. We will also explore the biochemical function of XopE35 and determine its key functional domains and active sites, and thereby understanding how XopE35 functions to modulate both JA and GA signaling. The study aims to reveal novel molecular mechanisms underlying the immunosuppression activity of the effector XopE35, which might lay a solid foundation for molecular design resistance breeding.

稻生黄单胞菌条斑致病变种引起的细菌性条斑病是水稻的重要病害，其分泌的效应蛋白在致病性中发挥关键作用。明确效应蛋白参与条斑病菌致病性的分子机理有助于制定高效防控该病害的新策略。前期研究发现条斑病菌效应蛋白XopE35在黄单胞菌中普遍存在，并能抑制植物免疫反应。初步结果还显示，该蛋白同时与植物激素茉莉酸（JA）和赤霉素（GA）信号通路中关键调控因子JAZ与SLR1蛋白互作。本项目将利用免疫共沉淀等技术对候选互作组合进行验证，研究XopE35在半离体与活体的条件下对靶标蛋白功能和稳定性的影响；并以xopE35转基因水稻为材料，通过全基因组表达谱分析确定XopE35对水稻JA与GA信号间交互作用的影响；探索XopE35的生化功能及其关键的功能域与活性位点，明确该蛋白作用于JA和GA信号通路的方式，厘清其调控植物免疫的新机理，完善水稻抗病调控网络。研究结果可望为水稻抗病分子设计育种提供重要基础。

稻生黄单胞菌条斑病菌致病变种Xanthomonas oryzae pv. oryzicola (Xoc)引起的细菌性条斑病是水稻上最重要的细菌病害之一，其分泌的效应蛋白在致病性中发挥关键作用。明确效应蛋白参与条斑病菌致病性的分子机理有助于制定高效防控该病害的新策略。条斑病菌效应蛋白XopE35（重新命名为XopC2）在黄单胞菌中普遍存在，系统进化树分析显示，XopC2的同源蛋白在多个植物病原细菌中广泛存在，比如Xanthomonas、Ralstonia、Acidovorax等，但其生化功能和发挥毒性功能的分子机制尚不清楚。本研究首先明确了XopC2在条斑病菌对水稻致病中的毒性功能。随后，揭示了XopC2属于一类新型的蛋白激酶家族成员。Xoc在侵染过程中分泌XopC2效应蛋白能够促进水稻的茉莉酸信号通路的激活，从而抑制水稻叶片上的气孔免疫，有助于细菌侵入叶片内部组织。第三，研究发现XopC2磷酸化OSK1蛋白，OSK1连接Cullin1和F-box蛋白OsCOI1b，在SCFOsCOI1b泛素连接酶复合体中起桥梁作用。进一步通过体外和体内实验证实XopC2能磷酸化OSK1蛋白第53位丝氨酸残基，该修饰促进了OSK1和OsCOI1b的结合以及SCFOsCOI1b复合体介导的OsJAZ9蛋白泛素化修饰和降解。此外，OSK1S53D与OsCOI1b结合亲和力增强依赖于OsCOI1b中第13位精氨酸残基。最后，表达Ser53模拟磷酸化突变体蛋白OSK1S53D的转基因水稻在外源茉莉酸甲酯处理下，JA下游响应基因表达上调增强，并且转基因水稻对Xoc的抗病性显著降低。本项目揭示了一类新型蛋白激酶家族及其生化功能，研究成果为后续水稻细条斑病抗病育种提供理论基础与新的思路。