Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain
Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain
复制标题
DOI：
10.1111/j.1365-313x.2009.04081.x
复制
发表时间：
2010-02-01
期刊：
影响因子：
7.2
通讯作者：
Ohme-Takagi, Masaru
中科院分区：
文献类型：
--
作者：
Matsui, Kyoko;Ohme-Takagi, Masaru
许多调节因子的活性涉及与其他蛋白质的相互作用。我们在这里证明了ERF相关的两亲性抑制(EAR)基序抑制域(SRDX)可以通过蛋白质-蛋白质相互作用介导的反转录作用将转录复合体转化为阻遏因子，并表明SRDX的反转录抑制活性可以用于检测这种蛋白质-蛋白质相互作用。当我们将与转录因子相互作用的蛋白质与SRDX融合并在植物细胞中与转录因子共表达时，作为转录因子靶标的基因的表达被反式抑制。我们使用FOS和JUN作为模型系统，并使用两个MADS盒植物蛋白pistillata和APETALA3来展示SRDX的反式抑制活性，这两个蛋白是已知形成异二聚体的。此外，与SRDX融合的表达TTG1的转基因植株表现出与ttg1突变体相似的表型，TTG1是一种WD40蛋白，与bHLH转录因子相互作用，并利用我们的系统检测了与bHLH蛋白相互作用所需的TTG1区域。我们还利用该系统分析了一个可能整合到转录复合体中的蛋白质因子，并通过比较35S：AtPWP2-SRDX和嵌合抑制物诱导的表型，鉴定了一个与AtTBP1相互作用的拟南芥WD40蛋白PWP2(AtPWP2)。我们的结果表明，SRDX介导的转录抑制可用于检测和确认植物中蛋白质-蛋白质的相互作用，并可用于识别形成转录蛋白质复合体的因素。
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.