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

PAPM EAGER：使用新颖的无克隆测序方法来发现宿主-微生物蛋白质-蛋白质相互作用

• 批准号: 1650227
• 负责人: Joseph Ecker
• 金额: $ 30万
• 依托单位: The Salk Institute for Biological Studies
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1650227&HistoricalAwards=false
• 关键词: PAPM; EAGER; Using; novel; clone

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.

该项目旨在开发一种简便的方法来挖掘植物-微生物相互作用，以获得影响植物性能的微生物因素的关键分子靶标。植物和微生物在从共生到致病的相互作用中相互传递分子。植物生物学家开始在分子水平上认识到这些相互作用的连续性。目前大多数的实验范例集中在病原微生物或共生微生物与特定植物的单一关联上。这些系统是高度发达的，具有各种分子和遗传研究工具包。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相互作用，在实验上还没有得到充分的开发。 在这里，将开发一种方法，可以作为一个通用的工具来了解蛋白质-蛋白质相互作用在任何植物-微生物相互作用。该项目预计将对植物及其微生物群之间的动态和密切相互作用产生原创见解。该方法随后可以作为植物微生物群功能研究的一个组成部分广泛部署，从而广泛影响植物科学研究。该项目将为本科生、博士生和博士后提供跨学科培训，并向高中生和本科生推广。待开发的方法可以作为一个通用的工具，以了解蛋白质-蛋白质相互作用在任何植物-微生物相互作用。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，研究界将很容易获得和利用。 这些数据将通过一个精心策划的PPIN数据库公开提供。该方法是一种名为ProCREate的高通量酵母双杂交（Y2 H）系统，其利用Cre-lox重组使蛋白质能够诱导Cre蛋白酶的报告基因表达，并随后诱导含有突变型loxP序列的质粒的Cre-recombination。每个蛋白质的相应编码序列的不可逆双突变loxP连接允许使用Illumina配对末端测序和生物信息学分析鉴定蛋白质相互作用。