PAPM EAGER: Using novel, clone-free sequencing methods to discover host-microbe protein-protein interactions

该项目旨在开发一种简便的方法来挖掘影响植物表现的微生物因子的关键分子靶标的植物-微生物相互作用。在从共生到致病的相互作用中，植物和微生物相互运输分子。植物生物学家开始在分子水平上欣赏这些相互作用的连续体。目前的大多数实验范式都集中在病原微生物或共生微生物与特定植物的单一联系上。这些系统高度发达，以各种用于研究的分子和遗传工具包为特色。然而，许多重要的植物-微生物相互作用，特别是那些导致毁灭性疾病的植物-微生物相互作用，在实验上是不发达的。这里将开发一种方法，它可以作为一种通用工具来理解任何植物-微生物相互作用中的蛋白质-蛋白质相互作用。预计该项目将对植物与其微生物区系之间的动态和密切相互作用产生原创性见解。该方法随后可以作为植物微生物区系功能研究的一个组成部分广泛应用，从而广泛影响植物科学研究。该项目将为本科生、博士和博士后提供跨学科培训，并向高中和本科生提供外联服务。将开发的方法可以作为一种通用工具来理解任何植物-微生物相互作用中的蛋白质-蛋白质相互作用。如果成功，该项目将提供一个前所未有的植物微生物蛋白质相互作用组信息来源，研究界将很容易地访问和利用这些信息。这些数据将通过一个经过管理的PPIN数据库公开提供。该方法是一种高通量酵母双杂交(Y2H)系统，称为PROCREATE，它利用Cre-lox重组使蛋白质能够诱导Cre重组酶的报告基因表达，并随后Cre-重组含有突变的loxP序列的质粒的Cre-重组。每种蛋白质相应编码序列的不可逆双突变loxP连锁允许使用Illumina配对末端测序和生物信息学分析来鉴定蛋白质相互作用。

This project seeks to develop a facile method to mine plant-microbe interactions for key molecular targets of microbial factors that influence plant performance. Plants and microbes traffic molecules to each other during interactions that range from symbiotic to pathogenic. Plant biologists are beginning to appreciate the continuum of these interactions at the molecular level. Most current experimental paradigms center on pathogenic or symbiotic microbes in mono-association with a specific plant. These systems are highly developed and feature various molecular and genetic tool kits for research. However, many important plant-microbe interactions, especially those causing devastating diseases, are experimentally under-developed. Here a method will be developed that can serve as a general tool to understand protein-protein interactions in any plant-microbe interaction. The project is expected to produce original insights into the dynamic and intimate interactions between plants and their microbiota. The method could be subsequently widely deployed as an integral part of functional study of plant microbiota, thereby broadly impacting plant science research. The project will provide interdisciplinary training for undergraduates, doctoral and post-doctoral as well as outreach to high school and undergraduate students. The method to be developed can serve as a general tool to understand protein-protein interactions in any plant-microbe interaction. If successful, the project will provide an unprecedented source of plant microbe protein interactome information that will be easily accessed and utilized by the research community. The data will be made publicly available via a curated PPIN database. The method is a high-throughput yeast two-hybrid (Y2H) system named ProCREate which exploits Cre-lox recombination to enable proteins to induce reporter gene expression of Cre Recombinase and subsequent Cre-recombination of plasmids containing mutant loxP sequences. The irreversible double mutant loxP linkage of each protein's corresponding coding sequence allows the identification of protein interactions using Illumina paired-end sequencing and bioinformatics analysis.