Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain.
The activities of many regulatory factors involve interactions with other proteins. We demonstrate here that the ERF-associated amphiphilic repression (EAR) motif repression domain (SRDX) can convert a transcriptional complex into a repressor via transrepression that is mediated by protein-protein interactions and show that transrepressive activity of SRDX can be used to detect such protein-protein interactions. When we fused a protein that interacts with a transcription factor with SRDX and co-expressed the product with the transcription factor in plant cells, the expression of genes that are targets of the transcription factor was suppressed by transrepression. We demonstrated the transrepressive activity of SRDX using FOS and JUN as a model system and used two MADS box plant proteins, PISTILLATA and APETALA3, which are known to form heterodimers. Furthermore, the transgenic plants that expressed TTG1, which is a WD40 protein and interacts with bHLH transcription factors, fused to SRDX exhibited a phenotype similar to ttg1 mutants by transrepression and the regions of TTG1 required for interaction to the bHLH protein were detected using our system. We also used this system to analyse a protein factor that might be incorporated into a transcriptional complex and identified an Arabidopsis WD40 protein PWP2 (AtPWP2) interacting with AtTBP1 through comparison of phenotypes induced by 35S:AtPWP2-SRDX with that induced by the chimeric repressor. Our results indicate that the transrepression mediated by SRDX can be used to detect and confirm protein-protein interactions in plants and should be useful in identifying factors that form transcriptional protein complexes.
Matsui
Kyoko
K
Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8562, Japan.
Ohme-Takagi
Masaru
M
D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D018749:RNA, Plant / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D029681:Arabidopsis Proteins; D018749:RNA, Plant; C121067:TTG1 protein, Arabidopsis; D014157:Transcription Factors