Plant Immunity pp 181-194 | Cite as
Purification of Effector–Target Protein Complexes via Transient Expression in Nicotiana benthamiana
- 30 Citations
- 3k Downloads
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.
Key words
Effectors Co-immunoprecipitation Oomycetes Phytophthora Mass spectrometry Target complexes Plant–microbe interactionNotes
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.
References
- 1.Dangl, J. and Jones, J. D. G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833.PubMedCrossRefGoogle Scholar
- 2.Jones, J. D. G. and Dangl, J. (2006) The plant immune system. Nature 444, 323–329.PubMedCrossRefGoogle Scholar
- 3.Kamoun, S. (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu. Rev. Phytopathol. 44, 41–60.PubMedCrossRefGoogle Scholar
- 4.Kamoun, S. (2007) Groovy times: filamentous pathogen effectors revealed. Curr. Opin. Plant Biol. 10, 358–365.PubMedCrossRefGoogle Scholar
- 5.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.PubMedCrossRefGoogle Scholar
- 6.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.PubMedCrossRefGoogle Scholar
- 7.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.PubMedCrossRefGoogle Scholar
- 8.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.PubMedCrossRefGoogle Scholar
- 9.Lindbo, J. (2007) TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol. 145, 1232–1240.PubMedCrossRefGoogle Scholar
- 10.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.PubMedCrossRefGoogle Scholar
- 11.Hellens, R., Mullineaux, P., and Klee, H. (2000) Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451.PubMedCrossRefGoogle Scholar