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

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Plant Fungal Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 835))

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 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.

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Acknowledgments

The author thanks Dr. Timothy Friesen and Dr. Melvin Bolton for reviewing the manuscript. All yeast two-hybrid work performed in the author’s laboratory is funded by the Agricultural Research Service, the United States Department of Agriculture under the CRIS project 5442-21000-033-00D.

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Authors and Affiliations

  1. Cereal Crops Research Unit, Northern Crop Science Laboratory, USDA-ARS, Fargo, ND, USA

    Shunwen Lu

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  1. Shunwen Lu
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Correspondence to Shunwen Lu .

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Editors and Affiliations

  1. Northern Crops Science Laboratory, USDA-ARS, North 18th Street 1307, Fargo, 58105-5677, North Dakota, USA

    Melvin D. Bolton

  2. Lab. Phytopathology, Wageningen University, Droevendaalsesteeg 1, Wageningen, 6708 PB, Netherlands

    Bart P.H.J. Thomma

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Lu, S. (2012). Use of the Yeast Two-Hybrid System to Identify Targets of Fungal Effectors. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_11

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  • DOI: https://doi.org/10.1007/978-1-61779-501-5_11

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-500-8

  • Online ISBN: 978-1-61779-501-5

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