Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain

Abstract

SUMMARYThe 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.

Transcriptional control of aspartate kinase expression during darkness and sugar depletion in Arabidopsis: involvement of bZIP transcription factors

“…Ehlert et al (2006) reported that in both yeast and plant cells similar bZIP-TF heterodimeric interactions occur. Previously, the SRDX-repressor motif was shown to interfere with the function of different transcription activators (Hiratsu et al 2003;Matsui and Ohme-Takagi 2010). However, its ability to antagonize bZIP repressors has only been shown recently, when constitutively overexpressed bZIP1-SRDX could release bZIP1 (belongs to the S group, Jakoby et al 2002) repression of seedling development under sugar shortage conditions (Kang et al 2010).…”

“…SRDX confers dominant transcriptional repression of target genes when fused to transcription activators (Hiratsu et al 2003(Hiratsu et al , 2004Fujita et al 2005). As yet the exact structural change exerted by the SRDX domain, when fused to various TFs, is not understood, nor its ability to trans-repress the activity of TFs when linked to proteins that specifically interact with these TFs (Kagale et al 2010;Matsui and Ohme-Takagi 2010). Our challenge was therefore to show that SRDX can antagonize the repression of AKs suggested to be exerted by DPBF4, ABI5 and other bZIP-TFs that share this repression task.…”

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine

“…The ability of such a domain to convert a transcriptional activator into a repressor has been widely shown by chimeric protein fusion experiments (Hiratsu et al, 2003;Matsui and Ohme-Takagi, 2010). Arabidopsis EAR domain-containing proteins show the DLNxxP or LxLxL sequence conservation pattern within the core sites (Kagale et al, 2010).…”

Members of the LATERAL ORGAN BOUNDARIES DOMAIN Transcription Factor Family Are Involved in the Regulation of Secondary Growth inPopulus

“…These typically employ fusion of LBD with the SRDX repressor domain, a short 12-amino acid motif that converts transcription factors to dominant repressors (Hiratsu et al, 2003). Transgenic plants expressing the fusion protein display a loss-of-function phenotype (Hiratsu et al, 2003;Matsui et al, 2004;Matsui and Ohme-Takagi, 2010). We therefore transformed WT-717 with a construct expressing LBD1 fused with the transcriptional repressor domain SRDX (Hiratsu et al, 2003).…”