Yeast two-hybrid assay to identify host-virus interactions

Stuart A. MacFarlane, Joachim F. Uhrig

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

    6 Citations (Scopus)

    Abstract

    The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.
    Original languageEnglish
    Title of host publicationPlant virology protocols
    Subtitle of host publicationfrom viral sequence to protein function
    EditorsGary D. Foster , I. Elisabeth Johansen, Yiguo Hong, Peter D. Nagy
    PublisherHumana Press
    Pages649-672
    Number of pages24
    ISBN (Electronic)9781597451024
    ISBN (Print)9781588298270
    DOIs
    Publication statusPublished - 2008

    Publication series

    NameMethods in molecular biology
    PublisherHumana Press
    Volume451
    ISSN (Print)1064-3745

    Fingerprint

    Two-Hybrid System Techniques
    Viruses
    Plant Viruses
    Proteins
    Yeasts
    Transcriptional Activation
    Plant Genome
    Plant Proteins
    DNA
    Viral Proteins
    Virus Diseases
    beta-Galactosidase
    Metabolic Networks and Pathways
    Life Cycle Stages
    Reporter Genes
    Histidine
    Leucine
    Libraries
    Mutation

    Keywords

    • DNA-Binding Proteins
    • Genetic Vectors
    • Host-Pathogen Interactions
    • Plant Diseases
    • Plant Viruses
    • Plants
    • Polymerase Chain Reaction
    • Saccharomyces cerevisiae
    • Saccharomyces cerevisiae Proteins
    • Transcription Factors
    • Two-Hybrid System Techniques

    Cite this

    MacFarlane, S. A., & Uhrig, J. F. (2008). Yeast two-hybrid assay to identify host-virus interactions. In G. D. Foster , I. E. Johansen, Y. Hong, & P. D. Nagy (Eds.), Plant virology protocols: from viral sequence to protein function (pp. 649-672). (Methods in molecular biology; Vol. 451). Humana Press. https://doi.org/10.1007/978-1-59745-102-4_44
    MacFarlane, Stuart A. ; Uhrig, Joachim F. / Yeast two-hybrid assay to identify host-virus interactions. Plant virology protocols: from viral sequence to protein function. editor / Gary D. Foster ; I. Elisabeth Johansen ; Yiguo Hong ; Peter D. Nagy. Humana Press, 2008. pp. 649-672 (Methods in molecular biology).
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    abstract = "The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.",
    keywords = "DNA-Binding Proteins, Genetic Vectors, Host-Pathogen Interactions, Plant Diseases, Plant Viruses, Plants, Polymerase Chain Reaction, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Two-Hybrid System Techniques",
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    MacFarlane, SA & Uhrig, JF 2008, Yeast two-hybrid assay to identify host-virus interactions. in GD Foster , IE Johansen, Y Hong & PD Nagy (eds), Plant virology protocols: from viral sequence to protein function. Methods in molecular biology, vol. 451, Humana Press, pp. 649-672. https://doi.org/10.1007/978-1-59745-102-4_44

    Yeast two-hybrid assay to identify host-virus interactions. / MacFarlane, Stuart A.; Uhrig, Joachim F.

    Plant virology protocols: from viral sequence to protein function. ed. / Gary D. Foster ; I. Elisabeth Johansen; Yiguo Hong; Peter D. Nagy. Humana Press, 2008. p. 649-672 (Methods in molecular biology; Vol. 451).

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

    TY - CHAP

    T1 - Yeast two-hybrid assay to identify host-virus interactions

    AU - MacFarlane, Stuart A.

    AU - Uhrig, Joachim F.

    PY - 2008

    Y1 - 2008

    N2 - The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.

    AB - The small size of most plant virus genomes and their very limited coding capacities requires that plant viruses are dependent on proteins expressed by the host plant for all stages of their life cycle. Identification of these host proteins is essential if we are to understand in any meaningful way the interactions that exist between virus and plant. A variety of methods are now available to isolate and study interacting proteins, however, the yeast two-hybrid (Y2H) assay system, which was one of the earliest mass analysis methods to be developed [Nature 340:245-246, 1989] remains one of the most popular and amenable approaches in current use. The Y2H method works by expressing two candidate interacting proteins together in the yeast cell. The (bait and prey) proteins under study are fused either to a promoter-specific DNA-binding domain or to a transcription activation domain. Interaction in the yeast nucleus between the bait and prey proteins brings the transcription activation and DNA-binding domains together so that they can initiate expression of a reporter gene. The reporter may be nonselective, such as the beta-galactosidase (LacZ) protein, or be selective by complementing a chromosomal mutation in a metabolic pathway for, for example, leucine or histidine biosynthesis. Individual bait proteins can be screened for interaction against a library of prey proteins, with any yeast colonies that grow on selective plates containing potential interacting partners. Using the Y2H system, a number of plant proteins interacting with viral proteins have been identified, recently, increasing our knowledge of the molecular basis of viral infection and host defense mechanisms.

    KW - DNA-Binding Proteins

    KW - Genetic Vectors

    KW - Host-Pathogen Interactions

    KW - Plant Diseases

    KW - Plant Viruses

    KW - Plants

    KW - Polymerase Chain Reaction

    KW - Saccharomyces cerevisiae

    KW - Saccharomyces cerevisiae Proteins

    KW - Transcription Factors

    KW - Two-Hybrid System Techniques

    U2 - 10.1007/978-1-59745-102-4_44

    DO - 10.1007/978-1-59745-102-4_44

    M3 - Chapter (peer-reviewed)

    SN - 9781588298270

    T3 - Methods in molecular biology

    SP - 649

    EP - 672

    BT - Plant virology protocols

    A2 - Foster , Gary D.

    A2 - Johansen, I. Elisabeth

    A2 - Hong, Yiguo

    A2 - Nagy, Peter D.

    PB - Humana Press

    ER -

    MacFarlane SA, Uhrig JF. Yeast two-hybrid assay to identify host-virus interactions. In Foster GD, Johansen IE, Hong Y, Nagy PD, editors, Plant virology protocols: from viral sequence to protein function. Humana Press. 2008. p. 649-672. (Methods in molecular biology). https://doi.org/10.1007/978-1-59745-102-4_44