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ARS Home » Midwest Area » St. Paul, Minnesota » Cereal Disease Lab » Research » Publications at this Location » Publication #395829
Research Project: Plant-Fungal Interactions and Host Resistance in Fusarium Head Blight of Barley and Wheat

Location: Cereal Disease Lab

Title: Bioinformatic analysis of yeast two-hybrid next-generation interaction screen data

Author
item VELÁSQUEZ-ZAPATA, VALERIA - Iowa State University
item Elmore, James - Mitch
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: Velásquez-Zapata, V., Elmore, J.M., Wise, R.P. 2023. Bioinformatic analysis of yeast two-hybrid next-generation interaction screen data. In: Mukhtar, S., editor. Protein-Protein Interactions, Methods and Protocols. Methods in Molecular Biology. 2690:223-239. https://doi.org/10.1007/978-1-0716-3327-4_19.
DOI: https://doi.org/10.1007/978-1-0716-3327-4_20

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 overview of the statistical analysis for Y2H-NGIS data, and an example outcome of this analysis: the interaction between the powdery mildew effector AVRA13 and the barley vesicle-mediated thylakoid membrane biogenesis protein, HvTHF1. This demonstrates the potential of an end-to-end screening that can be applied to any system of interest. In a complementary chapter, Elmore et al. provide an updated protocol for 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: We present the analytical basis of Y2H-NGIS (yeast two-hybrid next-generation-interaction screening) datasets. Different systems, libraries and experimental designs comprise the Y2H-NGIS technique. We summarize the analysis in several layers that comprise the characterization of baits and preys, quantification, and identification of true interactions for subsequent secondary validation. We present two software designed for this purpose, NGPINT and Y2H-SCORES which are used as front-end and back-end tools in the analysis. In this last section we explain how the Y2H-SCORES software can be used and adapted to analyze different datasets not only from Y2H-NGIS, but from other techniques ruled by similar biological principles.