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Yeast Two-Hybrid System for Dissecting the Rice MAPK Interactome

  • Raksha Singh
  • Sarmina Dangol
  • Nam-Soo JwaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)

Abstract

Protein–protein interactions are a preliminary but fundamental key to many biological systems. Identification of proteins that interact with particular bait not only contributes to a deeper understanding of bait protein function but also provides much information for the discovery of larger-scale interaction networks (interactome). Therefore, protein–protein interaction mapping is regarded as a widely accepted standardized functional genomics technique that provides comprehensive functional interpretation of previously uncharacterized proteins. A commonly used approach to detecting novel protein–protein interactions is the yeast two-hybrid system. In this chapter we describe in detail the protocols used to dissect the rice MAPK interactome, including the bait protein auto-activation test, identification of a rice MAPK interacting protein, confirmation of interaction by retransformation assay and characterization of the novel interacting protein.

Key words

Yeast two-hybrid Retransformation assay Auto-activation test Interactome Protein–protein interaction 

Notes

Acknowledgments

This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center; No. PJ008061), Rural Development Administration, Republic of Korea and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant number: 2013R1A1A2009269.

References

  1. 1.
    Yu J, Hu S, Wang J et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79–92PubMedCrossRefGoogle Scholar
  2. 2.
    Goff SA, Ricke D, Lan TH et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100PubMedCrossRefGoogle Scholar
  3. 3.
    Hamel LP, Nicole MC, Sritubtim S et al (2006) Ancient signals, comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci 11:192–198PubMedCrossRefGoogle Scholar
  4. 4.
    Reyna NS, Yang Y (2006) Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthegrisea infection. Mol Plant Microbe Interact 19:530–540PubMedGoogle Scholar
  5. 5.
    Arthur JS, Ley SC (2013) Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol 13:679–692PubMedGoogle Scholar
  6. 6.
    Singh R, Lee MO, Lee JE et al (2012) Rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system. Plant Physiol 160:477–487PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Singh R, Jwa NS (2013) The rice MAPKK–MAPK interactome, the biological significance of MAPK components in hormone signal transduction. Plant Cell Rep 32:923–931PubMedCrossRefGoogle Scholar
  8. 8.
    Seo YS, Chern M, Bartley LE et al (2011) Towards establishment of a rice stress response interactome. PLoS Genet 7:e1002020PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Ding X, Richter T, Chen M et al (2009) A rice kinase–protein interaction map. Plant Physiol 49:1478–1492CrossRefGoogle Scholar
  10. 10.
    Wankhede DP, Misra M, Singh P et al (2013) Rice mitogen activated protein kinase kinase and mitogen activated protein kinase interaction network revealed by in-silico docking and yeast two-hybrid approaches. PLoS One 30:e65011CrossRefGoogle Scholar
  11. 11.
    Agrawal GK, Iwahashi H, Rakwal R (2003) Rice MAPKs. Biochem Biophys Res Commun 302:171–180PubMedCrossRefGoogle Scholar
  12. 12.
    Song F, Goodman RM (2002) OsBIMK1, a rice MAP kinase gene involved in disease resistance responses. Planta 215:997–1005PubMedCrossRefGoogle Scholar
  13. 13.
    Agrawal GK, Agrawal SK, Shibato J et al (2003) Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation. Biochem Biophys Res Commun 300:775–783PubMedCrossRefGoogle Scholar
  14. 14.
    Xiong L, Yang Y (2003) Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell 15:745–759PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Cheong YH, Moon BC, Kim JK et al (2003) BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiol 132:1961–1972PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Kishi-Kaboshi M, Okada K, Kurimoto L et al (2010) A rice fungal MAMP-responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis. Plant J 63:599–612PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Xie G, Kato H, Imai R (2012) Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice. Biochem J 43:95–102CrossRefGoogle Scholar
  18. 18.
    Pandey A, Mann M (2000) Proteomics to study genes and genomes. Nature 405:837–846PubMedCrossRefGoogle Scholar
  19. 19.
    Uetz P, Hughes RE (2000) Systematic and large-scale two-hybrid screens. Curr Opin Microbiol 3:303–308PubMedCrossRefGoogle Scholar
  20. 20.
    ArabidopsisInteractome Mapping Consortium (2011) Evidence for network evolution in an Arabidopsis interactome map. Science 333:601–607CrossRefGoogle Scholar
  21. 21.
    Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246PubMedCrossRefGoogle Scholar
  22. 22.
    Walhout AJ, Boulton SJ, Vidal M (2000) Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm. Yeast 17:88–94PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Vidal M, Legrain P (1999) Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res 27:919–929PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Ito T, Chiba T, Ozawa R et al (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98:4569–4574PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Invitrogen (2005) ProQuest™ Two-Hybrid System. A sensitive method for detecting protein–protein interactions, catalog nos. PQ 10001-01 and PQ 10002-01, CA, USAGoogle Scholar
  26. 26.
    Clontech (2007) Matchmaker™ GAL4 two-hybrid system 3 and libraries user mannual. www.clontech.com/images/pt/PT3247-1.pdf
  27. 27.
    Vidal M (1997) The reverse two-hybrid system. The two-hybrid system. Oxford University Press, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Molecular BiologyCollege of Life Sciences, Sejong UniversitySeoulRepublic of Korea

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