RNAi and Plant Gene Function Analysis pp 109-127

Part of the Methods in Molecular Biology book series (MIMB, volume 744) | Cite as

Direct Transfer of Synthetic Double-Stranded RNA into Protoplasts of Arabidopsis thaliana

  • Ha-il Jung
  • Zhiyang Zhai
  • Olena K. Vatamaniuk
Protocol

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.

Key words

In vitro-synthesized dsRNA protoplasts protoplast viability assays RNAi 

References

  1. 1.
    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.CrossRefGoogle Scholar
  2. 2.
    Baulcombe, D. (2004) RNA silencing in plants. Nature 431, 356–363.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    Waterhouse, P. M. and Helliwell, C. A. (2003) Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. Genet. 4, 29–38.PubMedCrossRefGoogle Scholar
  8. 8.
    Zamore, P. D. (2001) RNA interference: listening to the sound of silence. Nat. Struct. Biol. 8, 746–750.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    Schwab, R., Ossowski, S., Warthmann, N., and Weigel, D. (2010) Directed gene silencing with artificial microRNAs. Methods Mol. Biol. 592, 71–88.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., and Liu, Y. (2003) Virus-induced gene silencing. Methods Mol. Biol. 236, 287–294.PubMedGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.Google Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    Heinemann, U. and Saenger, W. (1983) Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis. J. Biomol. Struct. Dyn. 1, 523–538.PubMedGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Udvardi, M. K., Czechowski, T., and Scheible, W.-R. (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20, 1736–1737.PubMedCrossRefGoogle Scholar
  23. 23.
    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.PubMedCrossRefGoogle Scholar
  24. 24.
    Salt, D. E. and Rauser, W. E. (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol. 107, 1293–1301.PubMedGoogle Scholar
  25. 25.
    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.PubMedCrossRefGoogle Scholar
  26. 26.
    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.PubMedCrossRefGoogle Scholar
  27. 27.
    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.PubMedCrossRefGoogle Scholar
  28. 28.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ha-il Jung
    • 1
  • Zhiyang Zhai
    • 1
  • Olena K. Vatamaniuk
    • 1
  1. 1.Department of Crop and Soil SciencesCornell UniversityIthacaUSA

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