稻瘟病菌多靶点效应蛋白（MAX-effectors）的人工设计及其与水稻受体互作的结构基础

Making full use of plant innate immune system to resist the pathogen invasion has potential application prospect for promoting sustainable development of green agriculture. Rice blast fungus (Magnaporthe oryzae) secretes a family of sequence-unrelated but structurally conserved effectors known as the MAX-effectors (M. Avrs and ToxB like). These MAX-effectors bind to structural conserved receptors (HMA-ID) by regulating the core binding interface, key residues and intermolecular interactions to trigger rice immune responses. Therefore, it is of great significance for enhancing host resistance by artificial engineering of MAX-effectors with multiple targets to bind to receptors..In this proposal, we will focus on the crystal structures of the rice receptors in complex with the target effectors (HMA-ID/MAX-effectors), and analyze the key residues affecting interaction. The following studies are proposed in this project: (1) the artificial design and functional analysis of multi-target effector proteins interacting with receptors; (2) the determination of structural basis of the interaction between multi-target effector proteins and receptors; (3) the investigation of biological activity in inducing host resistance of the key domain of multi-target effector. The results will fully activate the the rice innate immune system to resist pathogens, and will provide candidate materials and theoretical basis for the development of new environment-friendly plant immunity inducer with broad-spectrum resistance to rice blast.

充分利用植物天然免疫系统抵御病原入侵，对推进绿色农业可持续发展具有潜在应用前景。稻瘟病菌分泌一类序列一致性低、结构同源性高的效应因子（MAX-effectors），通过调整互作核心界面、关键残基及分子间互作力等，与结构保守的受体（HMA-ID）结合，诱导水稻抗性。因此，针对这类效应蛋白开展多靶点人工设计，对增强受体识别、激活寄主抗性具有重要意义。.本项目以已报道的水稻受体与稻瘟病菌效应蛋白（HMA-ID/MAX-effectors）复合物晶体结构为研究对象，分析影响效应蛋白互作的关键残基，并开展以下研究：（1）多靶点效应蛋白的人工设计与功能分析；（2）解析多靶点效应蛋白与受体互作的结构基础；（3）分析多靶点效应蛋白关键区域及其诱抗活性。这些研究有助于充分激发水稻天然免疫系统抵御病原菌的能力，为基于多靶点效应蛋白开发环境友好、广谱诱导寄主抗稻瘟病的新型植物免疫诱抗剂提供候选材料和理论基础。

稻瘟病菌分泌的一类效应因子（MAX-effectors）能够与水稻结构保守的受体（HMA-ID）结合诱导水稻抗性。针对这类效应蛋白开展多靶点人工设计，对增强受体识别、激活寄主抗性具有重要意义。本研究基于HMA-ID/MAX-effectors复合物晶体结构，以能够识别RGA5A_S和Pik-HMA 多个受体的AVR-PikD为研究对象，设计了8突变体AVR-PikD-M。利用原核表达系统获得RGA5A_S、Pikm-HMA、Piks-HMA和AVR1-CO39融合蛋白。通过Y2H、ITC和BiFc等技术筛选到突变体AVR-PikD(41-55)可在体外条件下与RGA5A_S、Pik-HMA形成稳定的复合物；采用坐滴气相扩散法获得RGA5A_S晶体，通过大规模晶体条件筛选并结合晶种RGA5A_S接种，尚未获得AVR-PikD-M/HMA-ID复合物晶体。蛋白-多肽docking建立了RGA5A_S/AVR-PikD(41-55)复合物结构，在此基础上，通过Y2H等验证上述推测，明确了AVR-PikD(41-55)Phe44His46Gly48是影响互作的关键残基；多肽AVR-PikD（41-55）进入寄主细胞是发挥其诱抗作用的关键环节，纳米载体可以作为农药助剂，提高农药分子的附着力和利用率，促进药剂被植物体吸收；本研究已获得纳米载体SPc/AVR-PikD（41-55）复合物。该研究为进一步推进纳米载体提升稻瘟病菌效应蛋白多肽AVR-PikD(41-55)进入寄主细胞诱导水稻抗稻瘟病研发提供重要基础。