Mining Ser hydrolase activities from the Pseudomonas-Arabidopsis interaction

植物与病原体的相互作用包括一个复杂的拮抗相互作用网络，其中对手的蛋白质活性由翻译后修饰、抑制剂和激活剂操纵。这种功能性蛋白质组学信息不能用传统的转录组学和蛋白质组学方法显示。基于活性的蛋白质分析（ABPP）是一项强大而独特的新技术，它使用基于活性的探针以机制依赖的方式与活性位点残基反应，显示蛋白质组的活性层。本项目提案旨在利用三功能氟膦酸盐（TriFP）探针鉴定丝氨酸水解酶（SHs）的差异活性。TriFP可以标记拟南芥基因组编码的600多种SHs，包括许多与免疫有关的SHs，如GDSL脂肪酶、EDS1、PAD4、甲基酯酶和枯草酶。初步实验揭示了丁香假单胞菌感染后SH的几种差异活性，目的是通过生物化学和反向遗传学来鉴定这些差异活性和其他差异SH活性，并研究它们在相容和不相容相互作用中的作用。

Plant-pathogen interactions comprise a complex network of antagonistic interactions where protein activities of the opponent are manipulated by post-translational modifications, inhibitors and activators. This functional proteomic information can not be displayed with traditional transcriptomic and proteomic approaches. Activity-based protein profiling (ABPP) is a robust and unique, novel technology that displays the activity layer of the proteome using activity-based probes that react with active site residues in a mechanism-dependent manner. This project proposal aims at identifying differential activities of serine hydrolases (SHs) using trifunctional fluorophosphonate (TriFP) probes. TriFP can label over 600 SHs encoded by the Arabidopsis genome, including numerous SHs involved in immunity such as GDSL lipases, EDS1, PAD4, methylesterases and subtilases. Preliminary experiments revealed several differential SH activities upon infection with Pseudomonas syringae, and the aim is to identify these and additional differential SH activities and investigate their role during compatible and incompatible interactions using biochemistry and reverse genetics.