Wheat Rust Diseases pp 85-98

Part of the Methods in Molecular Biology book series (MIMB, volume 1659) | Cite as

Protein–Protein Interaction Assays with Effector–GFP Fusions in Nicotiana benthamiana

  • Benjamin Petre
  • Joe Win
  • Frank L. H. Menke
  • Sophien Kamoun
Protocol

Abstract

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector–green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation–liquid chromatography–tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

Key words

Agroinfiltration Live-cell imaging Affinity chromatography DNA assembly Proteomics 

References

  1. 1.
    Rehman S, Gupta VK, Goyal AK (2016) Identification and functional analysis of secreted effectors from phytoparasitic nematodes. BMC Microbiol 16:48CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lo Presti L, Lanver D, Schweizer G, Tanaka S, Liang L, Tollot M, Zuccaro A, Reissmann S, Kahmann R (2015) Fungal effectors and plant susceptibility. Annu Rev Plant Biol 66:513–545CrossRefPubMedGoogle Scholar
  3. 3.
    Pais M, Win J, Yoshida K, Etherington GJ, Cano LM, Raffaele S, Banfield MJ, Jones A, Kamoun S, Saunders DG (2013) From pathogen genomes to host plant processes: the power of plant parasitic oomycetes. Genome Biol 14:211CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rodriguez PA, Bos JI (2013) Toward understanding the role of aphid effectors in plant infestation. Mol Plant-Microbe Interact 26:25–30CrossRefPubMedGoogle Scholar
  5. 5.
    Win J, Chaparro-Garcia A, Belhaj K, Saunders DG, Yoshida K, Dong S, Schornack S, Zipfel C, Robatzek S, Hogenhout SA, Kamoun S (2012) Effector biology of plant-associated organisms: concepts and perspectives. Cold Spring Harb Symp Quant Biol 77:235–247CrossRefPubMedGoogle Scholar
  6. 6.
    Petre B, Joly DL, Duplessis S (2014) Effector proteins of rust fungi. Front Plant Sci 5:416PubMedPubMedCentralGoogle Scholar
  7. 7.
    Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Mol Plant-Microbe Interact 25:1523–1530CrossRefPubMedGoogle Scholar
  8. 8.
    Goodin MM, Zaitlin D, Naidu RA, Lommel SA (2008) Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant-Microbe Interact 21:1015–1026CrossRefPubMedGoogle Scholar
  9. 9.
    Schornack S, van Damme M, Bozkurt TO, Cano LM, Smoker M, Thines M, Gaulin E, Kamoun S, Huitema E (2010) Ancient class of translocated oomycete effectors targets the host nucleus. Proc Natl Acad Sci U S A 107:17421–17326CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Petre B, Saunders DG, Sklenar J, Lorrain C, Krasileva KV, Win J, Duplessis S, Kamoun S (2016) Heterologous expression screens in Nicotiana benthamiana identify a candidate effector of the wheat yellow rust pathogen that associates with processing bodies. PLoS One 11:e0149035CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Petre B, Saunders DG, Sklenar J, Lorrain C, Win J, Duplessis S, Kamoun S (2015) Candidate effector proteins of the rust pathogen Melampsora larici-populina target diverse plant cell compartments. Mol Plant-Microbe Interact 28:689–700CrossRefPubMedGoogle Scholar
  12. 12.
    Win J, Kamoun S, Jones AM (2011) Purification of effector-target protein complexes via transient expression in Nicotiana benthamiana. Methods Mol Biol 712:1811–1894Google Scholar
  13. 13.
    Patron NJ (2016) Blueprints for green biotech: development and application of standards for plant synthetic biology. Biochem Soc Trans 44:702–708CrossRefPubMedGoogle Scholar
  14. 14.
    Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones JD, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff B, Miller B, Breakspear A, Radhakrishnan G, Delaux PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell TP, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger BR, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards KJ, Osbourn A, Haseloff J (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol 208:13–19CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Benjamin Petre
    • 1
  • Joe Win
    • 1
  • Frank L. H. Menke
    • 1
  • Sophien Kamoun
    • 1
  1. 1.The Sainsbury LaboratoryNorwichUK

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