Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants.

Abstract

We have developed a high-throughput yeast two-hybrid screening system (HTP-YTH) that incorporates yeast gap-repair cloning, multiple positive ( ADE2, HIS3, lacZ) and negative ( URA3-based) selection schemes to reduce the incidence of negative and false positive clones, and automation of laboratory procedures to increase throughput. This HTP-YTH system has been applied to the study of protein-protein interactions that are involved in rice defense signal transduction pathways. More than 100 genes involved in plant defense responses were selected from DuPont's rice expressed sequence tag (EST) databases as baits for HTP-YTH screening. Results from YTH screening of eight of these rice genes are presented in this paper. Not only have we identified known protein-protein interactions, but we have also discovered novel interactions, which may ultimately reveal the regulatory network of host defense signal transduction pathways. We have demonstrated that our HTP-YTH method can be used to map protein-protein interaction networks and signal transduction pathways in any system. In combination with other approaches, such efficient YTH screens can help us systemically to study the functions of known and unknown genes in the genomics era.