Plant Genomics

Lopez, Jessica, Mukhtar, M. Shahid, Jessica Lopez, M. Shahid Mukhtar

Wolfgang Busch

A tremendous asset to the analysis of protein-protein interactions is the yeast-2-hybrid (Y2H) method. The Y2H assay is a heterologous system that is expanding network biology knowledge via in vivo investigations of binary protein-protein interactions. Traditionally, the Y2H protocol entails the mating or co-transformation of yeast in solid agar media followed by visual analysis for diploid selection. Having played a key role in identifying protein-protein interactions for nearly three decades in a wide range of biological systems, the Y2H system assays the interaction between two proteins of interest which results in a reconstituted and/or activation of transcription factor allowing a reporter gene to be transcribed. Overall, the Y2H method takes advantage of two factors: (1) the auxotrophic yeast requires expression of the reporter gene to grow in media purposefully designed to lack one or more essential amino acids, and (2) the DNA-binding (DB) domain of transcription factor GAL4 is unable to initiate transcription unless it is physically associated with an activating domain (AD), which, together, DBs and ADs are fused to proteins of interest that must interact with each other to reconstitute the transcription factor and activate the reporter gene. The applications of Y2H are broad, entailing fields such as drug discovery, clinical trials for human disease including cancer and neurodegenerative disease, and extend even into synthetic biology applications and cellular engineering. This chapter begins with an introduction to the fundamental mechanics of Y2H utilizing a genetically engineered strain of yeast and proceeds with an in-depth look at the different types of Y2H and turn our focus particularly to the GAL4-based Y2H system to map protein-protein interactions. We will then provide a step-by-step protocol for the Y2H experimentation preceded by a materials listing while simultaneously including key notes throughout the entire experimental process of biological-mechanistic and historical understandings of the steps.