Abstract
Plant pathogens are responsible for enormous damage in natural and cultured ecosystems. One strategy most pathogenic organisms follow is the secretion of effector proteins that manipulate the host immune system to suppress defense responses. There is considerable interest in finding host targets of pathogen effectors as this helps to shape our understanding of how those proteins work in planta. The presented protocol describes a protein complex immunoprecipitation method aimed at verifying protein–protein interactions derived from protein complementation assays like Yeast-two-Hybrid.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Boch J, Bonas U (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol 48:419–436
Kim JG et al (2008) XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in xanthomonas-infected tomato leaves. Plant Cell 20:1915–1929
Shao F et al (2003) Cleavage of Arabidopsis PBS1 by a bacterial type III effector. Science 301:1230–1233
Nomura K et al (2006) A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313:220–223
Nomura K et al (2011) Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis. Proc Natl Acad Sci USA 108:10774–10779
Lee AHY et al (2012) A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion. PLoS Pathog 8:e1002523
Abramovitch RB et al (2006) Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity. Proc Natl Acad Sci USA 103:2851–2856
Gimenez-Ibanez S et al (2009) AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Curr Biol 19:423–429
van Damme M et al (2012) The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells. PLoS Pathog 8:e1002875
Bos J et al (2010) Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1. Proc Natl Acad Sci USA 107:9909–9914
Bozkurt TO et al (2011) Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface. Proc Natl Acad Sci USA 108:20832–20837
Saunders DGO et al (2012) Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance. Plant Cell 24:3420–3434
Mukhtar MS et al (2011) Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 333:596–601
Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–487
Gebert M, Dresselhaus T, Sprunck S (2008) F-actin organization and pollen tube tip growth in Arabidopsis are dependent on the gametophyte-specific Armadillo repeat protein ARO1. Plant Cell 20:2798–2814
Fabro G et al (2011) Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity. PLoS Pathog 7:e1002348
Braun P, Gingras A-C (2012) History of protein-protein interactions: from egg-white to complex networks. Proteomics 12:1478–1498
Field J et al (1988) Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165
Evan GI et al (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616
Brizzard B (2008) Epitope tagging. Biotechniques 44:693–695
Nakagawa T et al (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41
Earley KW et al (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629
Caillaud M-C et al (2012) Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility. Plant J 69:252–265
Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136
Rangan L, Vogel C, Srivastava A (2008) Analysis of context sequence surrounding translation initiation site from complete genome of model plants. Mol Biotechnol 39:207–213
Weigel D, Glazebrook J (2006) Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc 2006
Latijnhouwers M et al (2005) An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1. Plant J 44:459–470
Boevink PC, Birch PRJ, Whisson SC (2011) Imaging fluorescently tagged Phytophthora effector proteins inside infected plant tissue. Methods Mol Biol 712:195–209
Voinnet O et al (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956
Win J, Kamoun S, Jones AME (2011) Purification of effector-target protein complexes via transient expression in Nicotiana benthamiana. Methods Mol Biol 712:181–194
Zeiser E et al (2011) MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline. PLoS ONE 6:e20082
Villefranc JA, Amigo J, Lawson ND (2007) Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn 236:3077–3087
Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195
Nakagawa T et al (2007) Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71:2095–2100
Acknowledgments
DFAT and JS are funded by the UK Biotechnology and Biological Sciences Research Council grant BB/G015066/1 to JLB. The authors thank Mrs. Rachel Clewes and Mrs. Christina Payne for their technical support. The authors thank Dr. François Parcy (University Grenoble, France) for the BIFC vectors pBIFP1-4.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Steinbrenner, J., Eldridge, M., Tomé, D.F.A., Beynon, J.L. (2014). A Simple and Fast Protocol for the Protein Complex Immunoprecipitation (Co-IP) of Effector: Host Protein Complexes. In: Birch, P., Jones, J., Bos, J. (eds) Plant-Pathogen Interactions. Methods in Molecular Biology, vol 1127. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-986-4_16
Download citation
DOI: https://doi.org/10.1007/978-1-62703-986-4_16
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-985-7
Online ISBN: 978-1-62703-986-4
eBook Packages: Springer Protocols