Abstract
A crucial and often decisive test of a nuclear gene being involved in a given process is the complementation of mutants. Restoring the wild type phenotype by the wild type gene introduced into the mutant is a major piece of evidence for the function of this gene. We have developed a rapid and reliable method to complement protoplasts from plants with mutations in mitochondrial RNA editing with the respective wild type genes. The method furthermore allows testing the functionality of modified protein sequences without the need to make and grow transgenic plants, which is very time-consuming. We successfully employed this method for several nuclear-encoded genes involved in RNA editing at specific sites in mitochondria of Arabidopsis thaliana.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Giegé, P., and Brennicke, A. (1999) RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs. Proc Natl Acad Sci USA 96, 15324–15329.
Takenaka, M., Verbitskiy, D., van der Merwe, J. A., Zehrmann, A., and Brennicke, A. (2008) The process of RNA editing in plant mitochondria. Mitochondrion 8, 35–46.
Farré, J.-C., Leon, G., Jordana, X., and Araya, A. (2001) Cis recognition elements in plant mitochondrion RNA editing. Mol Cell Biol 21, 6731–6737.
Neuwirt, J., Takenaka, M., van der Merwe, J. A., and Brennicke, A. (2005) An in vitro RNA editing system from cauliflower mitochondria: editing site recognition parameters can vary in different plant species. RNA 11, 1563–1570.
van der Merwe, J. A., Takenaka, M., Neuwirt, J., Verbitskiy, D., and Brennicke, A. (2006) RNA editing sites in plant mitochondria can share cis-elements. FEBS Lett 580, 268–272.
Zehrmann, A., Verbitskiy, D., van der Merwe, J. A., Brennicke, A., Takenaka, M. (2009) A DYW-domain containing PPR-protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell 21, 558–567.
Zehrmann, A., van der Merwe, J. A., Verbitskiy, D., Brennicke, A., and Takenaka, M. (2008) Ecotype-specific variations in the extent of RNA editing in plant mitochondria. Mitochondrion 8, 319–327.
Kotera, E., Tasaka, M., and Shikanai, T. (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433, 326–330.
Okuda, K., Myouga, R., Motohashi, K., Shinozaki, K., and Shikanai, T. (2006) Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc Natl Acad Sci USA 104, 8178–1883.
Chateigner-Boutin, A.-L., Ramos-Vega, M., Guevara-Garcia, A., Andrés, C., Gutierrez-Nava, MdlL., Cantero, A., Delannoy, E., Jimenez, L. F., Lurin, C., Small, I. D., and León, P. (2008) CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts. Plant J 56, 590–602.
Yoo, S.-D., Cho, Y.-H., and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565–1572.
Arabidopsis Protocols (Martínez-Zapater, J. M., and Salinas, J. eds), (1998) Methods in Molecular Biology 82, Humana, Totowa, NJ.
Plant Gene Transfer and Expression Protocols (Jones, H. ed.), (1995) Methods in Molecular Biology 49, Humana, Totowa, NJ.
Picardi, E., Regina, T. M. R., Brennicke, A., and Quagliariello, C. (2007) REDIdb: the RNA editing database. Nucleic Acids Res 35, D173–D177.
Acknowledgments
We thank Dagmar Pruchner for excellent experimental help. This work was supported by a grant from the DFG to M.T.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Takenaka, M., Zehrmann, A. (2011). Complementation of Mutants in Plant Mitochondrial RNA Editing by Protoplast Transfection. In: Aphasizhev, R. (eds) RNA and DNA Editing. Methods in Molecular Biology, vol 718. Humana Press. https://doi.org/10.1007/978-1-61779-018-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-018-8_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-017-1
Online ISBN: 978-1-61779-018-8
eBook Packages: Springer Protocols