咯菌腈是一种广谱性的苯基吡咯类杀菌剂，对灰葡萄孢引起的灰霉病防治有特效，但其具体的作用方式和靶标尚不清楚。磷酸丙糖异构酶TPI是糖酵解过程的核心酶，对有效能量的生成至关重要。申请人前期研究发现，TPI氨基酸点突变导致灰葡萄孢对咯菌腈产生抗药性，靶向敲除TPI基因导致菌体对咯菌腈极度敏感，且上述敏感性的变化均依赖于III型组氨酸激酶Os1对HOG-MAPK信号途径的调控。为探究其作用机制，本项目拟从以下3个方面进行研究：1、构建TPI点突变载体，进行蛋白原核表达及纯化，测定TPI与咯菌腈之间的亲和性；2、测定咯菌腈对TPI酶活的影响；3、分析菌体受咯菌腈处理，TPI介导的糖酵解代谢物质变化对菌体药剂敏感性以及Os1功能和Hog1磷酸化的影响。本项目旨在阐明TPI调控灰葡萄孢对咯菌腈敏感性的分子机制，研究结果将为基于作用靶标的新农药分子设计提供理论基础，同时为咯菌腈的安全合理使用提供科学指导。

Fludioxonil is a broad-spectrum phenylpyrrole fungicide that exhibits specific activity on gray mold caused by Botrytis cinerea, but the mode of action ill defined and the direct drug target remains unknown. Triosephosphate isomerase (TPI) is a central enzyme of the glycolytic pathway that plays an important role in the generation of effective energy. Our previous study showed that point mutation in TPI conferred resistance of B. cinerea to fludioxonil, targeted deletion of TPI gene resulted in extreme sensitivity to fludioxonil, and these changes in sensitivity depended on the regulation of high osmolarity glycerol (HOG)-MAPK signaling pathway by the group III hybrid histidine kinase Os1. To explore the underlying mechanism of action, we are planning to do the research in the following aspects: 1. Construction of the TPI point mutation vector, protein expression and purification in vitro, and determine the binding affinity between TPI and fludioxonil; 2. Measurement of the effects of fludioxonil on enzyme activity of TPI; 3. Analysis of the effects of glycolytic metabolites changes mediated by TPI under fludioxonil treatment on drug sensitivity, the function of Os1 and Hog1 phosphorylation. The aim of this project is to clarify the molecular mechanism of TPI in regulating sensitivity of B. cinerea to fludioxonil. Results of this study will provide theoretical basis for the design of new pesticide based on target and scientific guidance for the use of fludioxonil safely and reasonably.