Direct Transfer of Synthetic Double-Stranded RNA into Protoplasts of Arabidopsis thaliana
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 wordsIn vitro-synthesized dsRNA protoplasts protoplast viability assays RNAi
- 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
- 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.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