Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectors

The yeast two-hybrid (Y2H) system is a binary method widely used to determine direct interactions between paired proteins. Although having certain limitations, this method has become one of the two main systemic tools (along with affinity purification/mass spectrometry) for interactome mapping in model organisms including yeast, Arabidopsis , and humans. It has also become the method of choice for investigating host–pathogen interactions in fungal pathosystems involving crop plants. This chapter describes general procedures to use the GAL4-based Y2H system for identification of host proteins that directly interact with proteinaceous fungal effectors, thus being their potential targets. The procedures described include cDNA library construction through in vivo recombination, library screening by yeast mating and cotransformation, as well as methods to analyze positive clones obtained from library screening. These procedures can also be adapted to confirmation of suspected interactions between characterized host and pathogen proteins or determination of interacting domains in partner proteins.