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Chapitre D'ouvrage Année : 2017

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

Benjamin Petre
Joe Win
Sophien Kamoun

Résumé

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.
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Dates et versions

hal-02548537 , version 1 (20-04-2020)

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Benjamin Petre, Joe Win, Frank Menke, Sophien Kamoun. Protein–Protein Interaction Assays with Effector–GFP Fusions in Nicotiana benthamiana. Wheat Rust Diseases, pp.85-98, 2017, ⟨10.1007/978-1-4939-7249-4_8⟩. ⟨hal-02548537⟩
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