Yeast Two-Hybrid Approaches to Dissecting the Plant Defense Response

Yeast Two-Hybrid Approaches Yeast Two-Hybrid Approaches to Dissecting the Plant Defense Response Mawsheng Chern, Todd Richter, and Pamela C. Ronald Summary We describe a reliable GAL4-based yeast two-hybrid system for identifying and isolat- ing clones encoding proteins interacting with a protein of interest. This two-hybrid system gives extremely low background and few false-positive clones, making it ideal for library screening purposes. We have successfully used it not only to isolate Arabidopsis NPR1- interactors from rice but also to pull out the rice NPR1 ortholog using one of the interactors as bait. Key Words: Yeast two-hybrid; GAL4; NPR1; defense. 1. Introduction The yeast two-hybrid approach has been widely used for library screening to identify and isolate genes encoding proteins that interact with a favorite protein. It has the capability of identifying and isolating desired clones in a relatively short time by allowing one to easily screen through tens of millions of clones. There are many two-hybrid systems available. Unfortunately, many of them may not be reliable; unacceptable backgrounds and troublesome false- positive results often are seen with many two-hybrid systems. Here, we describe a reliable GAL4-based yeast two-hybrid system that includes a bait plasmid vector derived from plasmid pPC86 (1), which is based on a CEN/ARS DNA replication system rather than the 2 P replication origin (see Note 1). This two- hybrid system is compatible with most two-hybrid libraries and gives extremely low background and few false-positive clones, making it ideal for library screening purposes (see Note 2). We have used it not only to isolate Arabidopsis NPR1-interactors from rice but also to pull out the rice ortholog of NPR1 by using one of the NPR1-interactors as bait to back-screen the rice From: Methods in Molecular Biology, vol. 354: Plant–Pathogen Interactions: Methods and Protocols Edited by: P. C. Ronald © Humana Press Inc., Totowa, NJ