ELUCIDATION OF HOST DEFENSE SIGNAL TRANSDUCTION PATHWAYS USING THE HIGH THROUGHPUT YEAST TWO-HYBRID SCREEN

1 Innovation Way, Newark, DE 19711

One of the fundamental biological questions is how proteins interact with each other at the cellular level. We have developed and implemented a high-throughput yeast two hybrid screening system (HTP-YTH) to study protein-protein interactions at the genomic scale. We have chosen the efficient homologous recombination-based gap-repair cloning method in place of traditional cloning method for the construction of large number of bait plasmids. Both the positive and negative selections have been utilized to increase the selection sensitivity as well as to reduce the number of false positives. Three different selection markers (two nutritional markers, ADE2 and HIS3, and the bacterial LacZ reporter gene) are incorporated for the positive selection at different stringencies. The URA3-based negative selection helps to eliminate the number of false positive clones during the screening. In addition, a number of laboratory automations have been introduced into the screening procedure. We are currently using this HTP-YTH system to study the protein-protein interactions that are involved in the rice defense signal transduction pathways. More than 130 genes that are found to be involved in plant defense responses have been selected from our EST databases and cloned as the baits. Large-scale HTP-YTH screening have been carried out and results will be presented.