XopZ-ORP1C互作调控水稻白叶枯病菌TAL效应子转运机制研究

Rice leaf blight is one of the most important rice diseases in the world and often causes great economic loss. Xoo (Xanthomonas oryzae pv. oryzae) TALEs (Transcription Activator-Like Effectors) and Non-TALEs (Non Transcription Activator-Like Effectors) have distinct pathologic functions, respectively. However, the collaboration mechanisms between them have not been reported before. In previous studies, we found that XopZ, a Non-TAL effector of rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo), may be involved in regulating the translocation of TAL effectors into rice cells. We used XopZ as bait and screened rice cDNA library by a conventional Y2H system, by using multiple controls, we clearly detected a direct interaction between XopZ and ORP1C (Oxysterol-binding protein-related protein in rice). In this project, we will use transgenic rice, protein interaction, effector and phospholipid translocation technology, for exploring the molecular mechanism for distribution of PI4P (phosphatidylinositol 4-phosphate) in rice cells, TAL effectors-PI4P interaction on the host cell membrane, and then regulating of the TAL effectors translocation in host cell that manipulated by XopZ-ORP1C interaction. All above-achieved results will elucidate the pathogenic mechanisms of bacterial leaf blight in rice varieties that manipulated by TAL and Non-TAL effectors collaboration for the first time. And will provide some scientific bases for the effective prevention of rice leaf blight under field conditions.

水稻白叶枯病是世界范围内重要的水稻病害之一，往往会造成巨大的经济损失。白叶枯病菌Non-TAL以及TAL效应子分别以独特的机制发挥截然不同的病理功能，但两类效应子能否在致病过程中发挥协同调控机制尚未见相关报道。本项目前期研究发现，白叶枯病菌PXO99菌株分泌的Non-TAL效应子XopZ可能参与调控TAL效应子向水稻胞内的转运。通过酵母双杂交筛库发现，XopZ与水稻氧化固醇结合蛋白ORP1C之间存在特异性互作。本项目拟在上述研究基础上，采用植物转基因、蛋白互作、效应子转运测定以及磷脂转运等技术，探究XopZ-ORP1C互作调控磷酯酰肌醇-4-磷酸（PI4P）在水稻胞内分布，促进TAL效应子在水稻细胞膜上与PI4P结合，进而调控其在水稻胞内转运的分子机制。研究结论将首次阐释Non-TAL和TAL效应子协同调控白叶枯病菌与水稻抗感性互作的分子机制，为制定有效的水稻白叶枯病防治策略提供科学依据。

水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae, Xoo）属于稻黄单胞菌种下致病变种，由其引起的白叶枯病是全世界范围内重要的水稻病害之一。Xoo侵染水稻后，通过III型分泌系统向水稻细胞中转运TAL效应子（Transcription Activator-Like Effectors）和Xop（Xanthomonas out protein）效应子蛋白。以往研究发现，Xop效应子会采取多样的生化手段攻击宿主PTI（PAMP-Triggered Immunity）通路重要组分，抑制宿主防卫反应以促进病原菌生长繁殖。而大多数Xop效应子彼此之间可能存在功能冗余，都能抑制寄主的PTI防卫反应。在PXO99菌株中，除了xopZ基因，单一敲除其他xop基因往往检测不到致病力的变化，我们推测XopZ不仅可以抑制寄主PTI反应，可能还存在其他未知的致病机制。在上述基础上，本研究首先构建xopZ无痕敲除突变体菌株∆xopZ及回补菌株∆xopZ/xopZ，通过水稻接种实验发现，xopZ基因缺失显著影响PXO99菌株在日本晴水稻上的致病力。利用酵母双杂交筛库发现，XopZ与水稻氧化固醇结合蛋白ORP1C之间存在特异性互作，并进一步通过免疫共沉淀和GST-Pulldown技术验证两者存在直接特异性互作。成功培育构建ORP1C基因敲除水稻株系；病原菌接种及相关分子实验表明， XopZ-ORP1C协同调控PXO99菌株在水稻宿主叶片上的致病力。而活性氧暴发和PTI标志基因表达检测测定发现，ORP1C未参与调控水稻PTI防卫反应。本项目研究结论为水稻白叶枯病害防治提供了新的理论依据，为水稻抗病育种提供了候选基因资源，同时丰富了植物病原细菌致病机制理论体系。