Abstract
Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector–reporter assays for functional analysis of transcription factors and detection of protein–protein interactions using the trans-repressive activity of SRDX repression domain are also described.
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References
Riechmann, J. L., Heard, J., Martin, G., Reuber, L., Jiang, C.-Z., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O. J., Samaha, R. R., Creelman, R., Pilgrim, M., Broun, P., Zhang, J. Z., Ghandhari, D., Sherman, B. K., and Yu, G.-L. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105–2110.
Mitsuda, N. and Ohme-Takagi, M. (2009) Functional analysis of transcription factors in Arabidopsis. Plant Cell Physiol. 50, 1232–1248.
Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M. (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739.
Matsui, K. and Ohme-Takagi, M. (2010) Detection of protein-protein interactions in plants using the transrepression activity of the EAR motif repression domain. Plant J. 61, 570–578.
Christensen, A. H., Sharrock, R. A., and Quail, P. H. (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18, 675–689.
Mitsuda, N. and Ohme-Takagi, M. (2008) NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. Plant J. 56, 768–778.
Hiratsu, K., Ohta, M., Matsui, K., and Ohme-Takagi, M. (2002) The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. FEBS Lett. 514, 351–354.
Acknowledgments
The authors express appreciation to Dr. Masaru Ohta, Dr. Keiichiro Hiratsu, Dr. Tomotsugu Koyama, Dr. Yoshimi Umemura, Dr. Masahito Shikata, and Dr. Akira Iwase who exerted great effort for the establishment and development of the protocols described in this chapter.
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Mitsuda, N. et al. (2011). CRES-T, An Effective Gene Silencing System Utilizing Chimeric Repressors. In: Yuan, L., Perry, S. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 754. Humana Press. https://doi.org/10.1007/978-1-61779-154-3_5
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DOI: https://doi.org/10.1007/978-1-61779-154-3_5
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