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

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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.