Location: Cereal Crops Research
Title: Use of The Yeast Two-Hybrid System to Identify Targets of Fungal Effectors Author
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: February 18, 2011
Publication Date: January 1, 2012
Citation: Lu, S. 2012. Use of the yeast two-hybrid system to identify targets of fungal effectors. In: M. Bolton & B. Thomma (eds), Plant Fungal Pathogens: Methods and Protocols, Methods in Molecular Biology 835:165-189. Technical Abstract: 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 human. 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 co-transformation as well as methods to analyze positive clones obtained from library screening. The application of these methods to a fungal pathosystem involving crop plants may lead to a better understanding of molecular mechanisms of host-pathogen interactions and possibly to discovery of novel targets for improving disease control strategies.