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ARS Home » Midwest Area » St. Paul, Minnesota » Cereal Disease Lab » Research » Publications at this Location » Publication #395830
Research Project: Plant-Fungal Interactions and Host Resistance in Fusarium Head Blight of Barley and Wheat

Location: Cereal Disease Lab

Title: Next-generation yeast two-hybrid screening to discover protein-protein interactions

Author
item Elmore, James - Mitch
item VELÁSQUEZ-ZAPATA, VALERIA - Iowa State University
item Wise, Roger

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 7/15/2023
Publication Date: 7/15/2023
Citation: Elmore, J.M., Velásquez-Zapata, V., Wise, R.P. 2023. Next-generation yeast two-hybrid screening to discover protein-protein interactions. In: Mukhtar, S.,editor. Protein-Protein Interactions, Methods and Protocols. Methods in Molecular Biology. 2690:205-222. https://doi.org/10.1007/978-1-0716-3327-4_19.
DOI: https://doi.org/10.1007/978-1-0716-3327-4_19

Interpretive Summary: Organisms respond to their environment through networks of interacting proteins and other biomolecules. In order to investigate these interacting proteins, many in vitro and in vivo techniques have been used. Among these, yeast two-hybrid (Y2H) has been adapted for use in combination with next generation sequencing (NGS) to approach protein-protein interactions on a genome-wide scale. The fusion of these two methods has been termed next-generation-interaction screening, abbreviated as Y2H-NGIS. In this chapter, we provide an updated step-by-step protocol for Y2H-NGIS that can be applied to any biological system of interest. In a complementary chapter, Velásquez-Zapata et al. provide a statistical framework for the analysis of protein-protein interactions from these high-throughput Y2H screens. Impact: These next generation methodologies provide a foundation for further research into the complex molecular components that control protein-protein interactions in crops.

Technical Abstract: Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for high-throughput sequencing libraries for identification, quantification, and ranking.