Modified Bimolecular Fluorescence Complementation Assay to Study the Inhibition of Transcription Complex Formation by JAZ Proteins

Methods Mol Biol. 2013;1011:187-97. doi: 10.1007/978-1-62703-414-2_15.

Abstract

The jasmonate (JA) ZIM-domain (JAZ) proteins of Arabidopsis thaliana repress JA signaling and negatively regulate the JA responses. Recently, JAZ proteins have been found to inhibit the transcriptional function of several transcription factors, among which the basic helix-loop-helix (bHLH) (GLABRA3 [GL3], ENHANCER OF GLABRA3 [EGL3], and TRANSPARENT TESTA8 [TT8]) and R2R3-MYB (GL1 and MYB75) that can interact with each other to form bHLH-MYB complexes and further control gene expression. The bimolecular fluorescence complementation (BiFC) assay is a widely used technique to study protein-protein interactions in living cells. Here we describe a modified BiFC experimental procedure to study the inhibition of the formation of the bHLH (GL3)-MYB (GL1) complex by JAZ proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / biosynthesis
  • Arabidopsis Proteins / genetics*
  • Basic Helix-Loop-Helix Transcription Factors / biosynthesis
  • Basic Helix-Loop-Helix Transcription Factors / genetics*
  • Cyclopentanes
  • DNA-Binding Proteins / biosynthesis
  • DNA-Binding Proteins / genetics*
  • Electroporation
  • Gene Expression
  • Gene Expression Regulation, Plant
  • Oxylipins
  • Protein Interaction Mapping*
  • Protein Multimerization
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism
  • RNA, Plant / genetics
  • RNA, Plant / isolation & purification
  • RNA, Plant / metabolism
  • Real-Time Polymerase Chain Reaction
  • Spectrometry, Fluorescence
  • Tobacco
  • Transcription, Genetic
  • Transformation, Genetic

Substances

  • Arabidopsis Proteins
  • Basic Helix-Loop-Helix Transcription Factors
  • Cyclopentanes
  • DNA-Binding Proteins
  • GL3 protein, Arabidopsis
  • Oxylipins
  • RNA, Messenger
  • RNA, Plant
  • GL1 protein, Arabidopsis
  • jasmonic acid