Abstract
Efficient gene silencing tools are extremely important for genomics and biotechnology applications. A number of aberrant RNA species induce silencing of homologous genes; however, only the inverted repeat RNA is widely utilized for targeted gene silencing in plants. Development of additional silencing constructs would not only provide an alternative technology for single targets, it may also serve as a simpler platform for multi-target silencing. The present study found that aberrantly terminated sense transcripts serve as efficient inducers of gene silencing. A construct initially designed to carry out homologous recombination into the Phytochrome A locus of Arabidopsis induced instead a high rate of Phytochrome A silencing, resulting in a strong mutant phenotype. Attempts to decipher the silencing determinant of the construct revealed that absence of a proper transcription termination signal contributed to the silencing efficiency of the construct. Furthermore, silencing of an additional gene, Phytochrome B, was conferred by a separate construct of a similar design. Proper placement of a transcription termination signal in the silencing construct abolished its silencing ability. Therefore, transcription of aberrantly terminated transcripts was deduced to be the cause of the elevated rate of silencing observed in transgenic lines. These transcripts could be produced either by direct transcription or read-through transcription. Since silencing resulted in strong mutant phenotypes in the majority of transgenic lines, terminator-less constructs are proposed to serve as efficient tools for knocking out endogenous targets.
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Acknowledgments
This study was supported by funds provided by Arkansas Bioscience Institute and Arkansas Division of Agriculture. We thank Dr. P. Quail for providing PHYA and PHYB clones.
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Communicated by A. Aguilera.
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Nicholson, S.J., Srivastava, V. Transgene constructs lacking transcription termination signal induce efficient silencing of endogenous targets in Arabidopsis. Mol Genet Genomics 282, 319–328 (2009). https://doi.org/10.1007/s00438-009-0467-1
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DOI: https://doi.org/10.1007/s00438-009-0467-1