Abstract
Double-stranded (ds) RNA interference (RNAi) is widely used as a reverse genetic approach for functional analysis of plant genes. Constitutive or transient RNAi effects in plants have been achieved via generating stable transformants expressing dsRNAs or artificial microRNAs (amiRNAs) in planta or by viral-induced gene silencing (VIGS). Although these tools provide outstanding resources for functional genomics, they require generation of vectors expressing dsRNAs or amiRNAs against targeted genes, transformation and propagation of transformed plants, or maintenance of multiple VIGS lines and thus impose time, labor, and space requirements. As we showed recently, these limitations can be circumvented by inducing RNAi effects in protoplasts via transfecting them with in vitro-synthesized dsRNAs. In this chapter we detail the procedure for transient gene silencing in protoplasts using synthetic dsRNAs and provide examples of approaches for subsequent functional analyses.
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References
Allen, R. S., Millgate, A. G., Chitty, J. A., Thisleton, J., Miller, J. A. C., et al. (2004) RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotech. 22, 1559–1566.
Baulcombe, D. (2004) RNA silencing in plants. Nature 431, 356–363.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Kennerdell, J. R. and Carthew, R. W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026.
Smith, N. A., Singh, S. P., Wang, M.-B., Stoutjesdijk, P. A., Green, A. G., et al. (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407, 319–320.
Vidali, L., Augustine, R. C., Kleinman, K. P., and Bezanilla, M. (2007) Profilin is essential for tip growth in the moss Physcomitrella patens. Plant Cell 19, 3705–3722.
Waterhouse, P. M. and Helliwell, C. A. (2003) Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. Genet. 4, 29–38.
Zamore, P. D. (2001) RNA interference: listening to the sound of silence. Nat. Struct. Biol. 8, 746–750.
Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, P., Yamamoto, Y. Y., et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190.
Schwab, R., Ossowski, S., Warthmann, N., and Weigel, D. (2010) Directed gene silencing with artificial microRNAs. Methods Mol. Biol. 592, 71–88.
Burch-Smith, T. M., Schiff, M., Liu, Y., and Dinesh-Kumar, S. P. (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142, 21–27.
Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., and Liu, Y. (2003) Virus-induced gene silencing. Methods Mol. Biol. 236, 287–294.
Lu, R., Martin-Hernandez, A. M., Peart, J. R., Malcuit, I., and Baulcombe, D. C. (2003) Virus-induced gene silencing in plants. Methods 30, 296–303.
Zhai, Z., Sooksa-nguan, T., and Vatamaniuk, O. K. (2009) Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts. Plant Physiol. 149, 642–652.
Sastry, S. S. and Ross, B. M. (1997) Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes. J. Biol. Chem. 272, 8644–8652.
Zhai, Z., Jung, H. I., and Vatamaniuk, O. K. (2009) Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J. Vis. Exp. doi: 10.3791/1149.
Rong, M., He, B., McAllister, W. T., and Durbin, R. K. (1998) Promoter specificity determinants of T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 95, 515–519.
Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc. Natl. Acad. Sci. USA 96, 7110–7115.
Karve, A., Rauh, B. L., Xia, X., Kandasamy, M., Meagher, R. B., et al. (2008) Expression and evolutionary features of the hexokinase gene family in Arabidopsis. Planta 228, 411–425.
Heinemann, U. and Saenger, W. (1983) Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis. J. Biomol. Struct. Dyn. 1, 523–538.
Remans, T., Smeets, K., Opdenakker, K., Mathijsen, D., Vangronsveld, J., et al. (2008) Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227, 1343–1349.
Udvardi, M. K., Czechowski, T., and Scheible, W.-R. (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20, 1736–1737.
Howden, R., Goldsbrough, P. B., Andersen, C. R., and Cobbett, C. S. (1995) Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol. 107, 1059–1066.
Salt, D. E. and Rauser, W. E. (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol. 107, 1293–1301.
Chen, A., Komives, E. A., and Schroeder, J. I. (2006) An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis. Plant Physiol. 141, 108–120.
Mendoza-Cozatl, D. G., Butko, E., Springer, F., Torpey, J. W., Komives, E. A., et al. (2008) Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation. Plant J. 54, 249–259.
Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides. J. Biol. Chem. 275, 31451–31459.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
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Jung, Hi., Zhai, Z., Vatamaniuk, O.K. (2011). Direct Transfer of Synthetic Double-Stranded RNA into Protoplasts of Arabidopsis thaliana . In: Kodama, H., Komamine, A. (eds) RNAi and Plant Gene Function Analysis. Methods in Molecular Biology, vol 744. Humana Press. https://doi.org/10.1007/978-1-61779-123-9_8
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DOI: https://doi.org/10.1007/978-1-61779-123-9_8
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