Yeast Two-Hybrid Assay to Identify Host–Virus Interactions

Abstract

The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.