Yeast Two-Hybrid System for Dissecting the Rice MAPK Interactome
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 wordsYeast two-hybrid Retransformation assay Auto-activation test Interactome Protein–protein interaction
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.
- 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.Clontech (2007) Matchmaker™ GAL4 two-hybrid system 3 and libraries user mannual. www.clontech.com/images/pt/PT3247-1.pdf
- 27.Vidal M (1997) The reverse two-hybrid system. The two-hybrid system. Oxford University Press, New YorkGoogle Scholar