Abstract
Effectors of plant pathogens play important roles in not only pathogenesis but also plant immunity. Plant pathogens use these effectors to manipulate host cells for colonization, and their activities likely influence the evolution of plant immune responses. Analyses of genome sequences revealed that oomycete pathogens, such as Phytophthora spp., possess hundreds of RXLR effectors that are thought to be delivered into the host cells and hence function inside the cells by interacting with the host protein complexes. This article describes a co-immunoprecipitation protocol aimed at identifying putative target complexes of the effectors by transiently overexpressing the tagged effectors in planta. The identification of the eluted protein complexes was achieved by LC-MS/MS mass spectrometry and peptide spectrum matching.
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 subscriptionsReferences
Dangl, J. and Jones, J. D. G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833.
Jones, J. D. G. and Dangl, J. (2006) The plant immune system. Nature 444, 323–329.
Kamoun, S. (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu. Rev. Phytopathol. 44, 41–60.
Kamoun, S. (2007) Groovy times: filamentous pathogen effectors revealed. Curr. Opin. Plant Biol. 10, 358–365.
Tyler, B. M., Tripathy, S., Zhang, X., Dehal, P., Jiang, R. H., et al. (2006) Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313, 1261–1266.
Win, J., Morgan, W., Bos, J., Krasileva, K. V., Cano, L. M., Chaparro-Garcia, A., Ammar, R., Staskawicz, B. J., and Kamoun, S. (2007) Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes. Plant Cell 19, 2349–2369.
Sacco, M. A., Mansoor, S., and Moffett, P. (2007) A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance. Plant J. 52, 82–93.
Monti, M., Cozzolino, M., Cozzolino, F., Vitiello, G., Tedesco, R., Flagiello, A., and Pucci, P. (2009) Puzzle of protein complexes in vivo: a present and future challenge for functional proteomics. Expert Rev. Proteomics 6, 159–169.
Lindbo, J. (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol. 145, 1232–1240.
Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using stagetips. Nat. Protoc. 2, 1896–1906.
Hellens, R., Mullineaux, P., and Klee, H. (2000) Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.
Acknowledgments
We thank John Lindbo (UC Davis) for providing us with the TRBO vector, and Liliya Serazetdinova for technical assistance. This work was supported by the Gatsby Charitable Foundation.
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
Win, J., Kamoun, S., Jones, A.M.E. (2011). Purification of Effector–Target Protein Complexes via Transient Expression in Nicotiana benthamiana . In: McDowell, J. (eds) Plant Immunity. Methods in Molecular Biology, vol 712. Humana Press. https://doi.org/10.1007/978-1-61737-998-7_15
Download citation
DOI: https://doi.org/10.1007/978-1-61737-998-7_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61737-997-0
Online ISBN: 978-1-61737-998-7
eBook Packages: Springer Protocols