抗霉枯草菌素触发植物诱导性系统抗性的作用受体及机制研究

芽孢杆菌等根际益生菌（PGPR）能诱导植物系统抗性（ISR），防治作物病害具有安全长效等优点，但其触发机制不清，互作理论不完善。前期证明筛于新疆棉花根际的枯草芽孢杆菌J-15可高效生防黄萎病，其环脂肽类代谢物-抗霉枯草菌素（Myco）能高效广谱拮抗真菌，还可激发JA/ET等信号通路，进而触发植物ISR，为研究环脂肽触发ISR提供了极佳的材料。项目拟构建Otyl-Myco亲和胶粒，借鉴亲和层析及Pull-D思路，筛钓Myco的植物结合蛋白。并以显著响应Myco的J A/ET、SA、IA、BR等信号通路上游AOS、ACS、ICS、IAOX、BIR等因子为指征筛定结合蛋白，通过对比目标蛋白过表达及沉默对Myco触发ISR的影响，验证其受体功能，并采用等温滴定量热及核磁共振等技术解析互作机制。以期明确Myco受体、揭示Myco及其他环脂肽触发植物ISR的机制，完善植物与根际微生物的互作理论体系。

Plant rhizosphere probiotics (PGPR) such as bacillus can induce systemic resistance (ISR). Using PGPR to control crop diseases has the advantages of safety and long-term effect. However, the mechanism of PGPR triggering plant ISR is not clear, leading to no mature theory to guide us to make efficient use of PGPR..In the previous study, the project team obtained a Bacillus subtilis J-15 which can effectively control crop Verticillium wilt, and it has been confirmed by experiments that the strain can produce a metabolite of cyclic lipopeptide, namely mycomycin (Myco). Myco can not only kill fungi such as Verticillium wilt, but also activate JA/ETH, MAPK and other signaling pathways, and further trigger plant ISR. Therefore, Myco is a good pesticide resource to improve plant disease resistance. Moreover, the previous research and experience provide a good experimental material for us to further explore the role of plant in recognizing Myco receptors, and also provide a solid foundation for revealing the mechanism of PGPR triggering plant ISR. In addition, we found that hydrophobic membrane or column can effectively immobilize Myco molecules. Based on the molecular structure and physicochemical properties of Myco, we want to screen the filling material (cellfinetm octyl) to make the binding protein by affinity "Octyl Myco" colloidal particles. The micro affinity chromatography column was made by using the colloidal particles as the packing. We can add the extracts of plant cell membrane components to the specific separation column and elute with high concentration of acetonitrile to obtain proteins that can bind with Myco. These candidates were incubated with Myco in vitro, and their binding was detected by LC-MS. Based on the information of bioinformatics and previous studies, the relationship between these identified candidate receptor molecules and the expression of AOS and other factors upstream of JA signaling pathway was analyzed.We will obtain Arabidopsis mutants of the candidate receptor genes and tested whether the mutants could respond to Myco induction. We can judge whether the mutants respond to the induction of Myco based on the previous transcriptome and metabolomic analysis of AOS and other factors in the upstream of JA and other signaling pathways regulated by Myco. Mutants that do not respond to Myco induction can be considered as key candidates for Myco receptors.The response of plants in these experiments to Myco was compared by overexpression of key candidate receptors, silencing, or complementation of mutants. Finally, we determined the receptor of Myco on the membrane of plant target cells..In addition, isothermal titration calorimetry and nuclear magnetic resonance (NMR) were used to detect the binding domain of Myco and specific receptor, and to explore the mechanism of interaction between Myco and specific receptor..The goal of this project is to identify the proteins involved in triggering plant ISR by Myco, and to reveal the mechanism of triggering plant ISR by cyclopeptides such as Myco, so as to lay a foundation for improving the theoretical system of interaction mechanism between plants and microorganisms.