When short peptides that contained the EAR motif were fused to activators of transcription, the resultant chimeric transcription factors acted as strong repressors and suppressed the expression of a reporter gene in the presence of another activator of transcription in transient expression assays in Arabidopsis, even if the activator contained the VP16 activation domain of herpes simplex virus ( Hiratsu et al., 2002 Ohta et al., 2001). We previously reported that the repression domains of the class II ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR (ERF) and TFIIIA-type zinc finger repressors of transcription that include SUPERMAN (SUP) contain the EAR (ERF-associated amphiphilic repression) motif ( Hiratsu et al., 2002 Ohta et al., 2001). Thus, repression domains that function in plant cells and can overcome the genetic redundancy of plant transcription factors are necessary if we are to utilize chimeric repressors or the identification of the biologic functions of plant transcription factors. Moreover, addition of a long peptide, such as En, which includes approximately 300 amino acids, might affect the integral properties of some plant transcription factors, such as protein–protein interactions and/or dimerization. This observation suggests that the mechanisms for the repression of transcription might differ between animals and plants. In fact, the KRAB repression domain acts as a transcriptional activator in transient expression assays in Arabidopsis when fused to the GAL4 DNA-binding domain (unpublished results). However, the repressive activity of animal repression domains has not been demonstrated clearly in plant cells. Such chimeric repressors have been shown to act dominantly in animals, and thus a similar strategy might be expected to facilitate the analysis of redundant transcription factors in plants. The repression domains from the Krüppel-associated box (KRAB) of the human estrogen receptor, Engrailed (En) of Drosophila or mSIN3 interaction domain (Sid) of humans, have been used effectively in mammalian cells ( Badiani et al., 1994 Beerli et al., 1998, 2000 de Haan et al., 2000 John et al., 1995). In animal cells, chimeric repressors, in which a DNA-binding domain or a transcription factor is fused to a repression domain, have been used for the targeted repression of the expression of genes of interest ( Badiani et al., 1994 Beerli et al., 1998, 2000 de Haan et al., 2000 John et al., 1995). Even when gene-knockout or antisense lines specific for a particular transcription factor can be isolated, such lines often fail to exhibit an informative phenotype that might provide some direct clue to the factor's function, for example, a loss-of-function phenotype ( Borevitz et al., 2000 Bouché and Bouchez, 2001 Meissner et al., 1999). This structural and functional redundancy of plant transcription factors often interferes with efforts to identify the functions of these factors. Plant genes are frequently duplicated, and many plant transcription factors form large families in which family members include strongly conserved DNA-binding domains ( Riechmann et al., 2000). However, the functions and target genes of most plant transcription factors remain to be characterized. In addition, the transcription factors encoded in plant genomes exhibit much greater variety than those in animals, and numerous plant-specific transcription factors have been recognized ( Riechmann et al., 2000). The genome of Arabidopsis contains more than twice as many genes for transcription factors as the genomes of animals with genomes of sizes similar to that of Arabidopsis ( Riechmann et al., 2000). Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors. This chimeric repressor silencing technology (CRES-T), exploiting the EAR-motif repression domain, is simple and effective and can overcome genetic redundancy. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors.
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