水稻细胞膜水通道蛋白质衔接细胞内外抗病性信号传导的结构基础

Hydrogen peroxide (H2O2) is a stable component of reactive oxygen species and its production in plants represents the successful recognition of pathogen infection and pathogen-associated molecular patterns (PAMPs). This production of H2O2 is typically apoplastic but is subsequently associated with intracellular immunity pathways that regulate disease resistance. Obviously, a cytological gap exists between H2O2 generation and functional performance, which remains a long-unanswered question. To address this question, we recently analyzed possible roles of plasma membrane intrinsic proteins (PIPs), which compose one of five major families of aquaporins in plants, in transport of extracellular H2O2 into intracellular spaces. The PIP family has 11 members in rice while PIP2;4, 2;5, and 2;7 are able to mediate the cytoplasmic import of H2O2 externally applied to yeast cultures. Thus, the three PIP2s are effective facilitators of H2O2 transport across the plasma membrane (PM). The most abundant translocation of H2O2 is provided by PIP2;4 owing to two pairs of distinct amino acid resides in transmembrane (TM) domains 2 and 3 of six TMs, which characterize all aquaporins..In connection with our previous studies, the present project is focused on resolution of the question how the induced apoplastic H2O2 is mechanically connected with intracellular immunity pathways. We will use specific fluorescent days to monitor the cytological dynamics of pathogen/PAMP-induced apoplastic H2O2 and determine the roles that PIP2;4, 2;5, and 2;7 play in cytoplasmic import of H2O2 across rice PMs. Whether the three PIP2s fulfill the H2O2 transport function in a redundant manner is to be demonstrated. As PIP2;4 is a predominant facilitator for H2O2 transport across PMs, two variants of PIP2;4 have been generated by residue replacements in pair at TM2 and TM3, respectively. PIP2;4 and its variants will be phosphorylated and dephosphorylated, respectively, and crystalized alone or together with H2O2, followed by X-ray diffraction. X-ray diffraction data will be collected at Sub-Ångstrom levels and analyzed to disclose the fine structure of PIP2;4-H2O2 interaction within the selectivity filter, and therefore to demonstrate the structural basis of the role that PIP2;4 plays in H2O2 transport. Topological movements of the selectivity filter and central channel, and for both functional structures prototropic and valent tautomerism will be detected to disclose how these atomic forces facilitate H2O2 transport by OsPIP2;4. Evidently, results obtained from these analyses will profoundly elucidate a previously unappreciated mechanism that underpins apo-cytoplastic signal transduction in immunity pathways. More significantly, the structural basis of PIP2;4-mediated H2O2 translocation will provide new insights into reoccurring structural themes of aquaporins in both gating and trafficking.

植物识别病菌侵染，迅速在质外体产生过氧化氢，随后启动细胞内免疫反应。针对细胞内外免疫信号如何传导的问题，我们探讨了水稻细胞质膜上11种水通道蛋白质(PIP)参与作用的可能性。在重组酵母细胞中，PIP2;4、2;5和2;7都能介导过氧化氢跨膜转运，其中PIP2;4作用最强，这由它底物选择与运输功能域附近两对氨基酸所决定。本项目拟研究其中的机制，测定水稻由病菌侵染诱导的过氧化氢跨膜运输动态与水稻免疫反应的关系，明确上述三种PIPs参与的作用及其功能冗余性调控方式。PIP2;4和在上述位点诱变的蛋白质经磷酸化与去磷酸化处理，单独结晶并与过氧化氢共结晶，经X-射线衍射，在亚埃水平上采集、分析数据，建立结构模型，解析PIP2;4底物选择与运输功能域拓扑结构位移与互变异构等因素对过氧化氢跨膜运输的调控作用。结果将揭示PIP介导过氧化氢跨膜运输的结构基础，阐明植物细胞内外免疫信号传导的联络机制。

本课题围绕水稻（Oryza sativa）细胞膜嵌入蛋白（plasma membrane intrinsic protein, PIP）家族的水通道蛋白（aquaporins, AQPs）参与免疫信号传导和抗病防卫反应调控的分子机制开展研究，揭示了OsPIP1;3和OsPIP2;2调控水稻“生长-防卫对抗性博弈关系（growth-defense tradeoffs）”的功能机理，表现为以下七个方面。一、病原物侵染或模式分子处理而诱导水稻在质外体产生H2O2，OsPIP2;2介导H2O2转入细胞质，这是水稻免疫信号传导在细胞内外联络的一个节点。二、OsPIP2;2介导MYB转录因子从细胞膜向细胞核转运，激活抗病防卫反应。三、拟南芥（Arabidopsis thaliana）高效运输H2O2的AtPIP2;4在底物运输中心通道上的3.7Å结构特征，与已知运输H2O分子的AQPs无明显差别。这说明，凡是能运输H2O的AQPs或可都能运输H2O2，H2O2可能以水溶状态进行运输。四、OsPIP1;3的底物不仅有H2O2，还有CO2；不仅能将病原物或模式分子诱导的水稻质外体H2O2转入细胞质，从而激活免疫信号传导和抗病防卫反应，而且还能将CO2从大气环境转入水稻细胞质，从而促进水稻叶片光合作用，结果导致籽粒产量显著提高。五、OsPIP2;2和OsPIP1;3可同时因应病菌或模式分子诱导的质外体H2O2浓度提高，合作增效，提高H2O2运输量，增强抗病防卫反应。六、OsPIP1;3识别病原细菌转位子蛋白Hpa1，OsPIP1;3-Hpa1互作可以促使病原细菌致病效应子从细菌细胞向水稻细胞转运，从而影响病菌毒性和白叶枯病严重程度。七、利用基因编辑等生物技术方法或分子识别干扰等生化一直方法，可以抑制OsPIP1;3-Hpa1识别，迫使OsPIP1;3在细菌效应子与CO2转运之间发生功能转换，进而打破生长-防卫对抗性的博弈关系，达到兼顾病害防治和保证或提高作物产量的目的。