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

Petre, Benjamin, Win, Joe, Menke, Frank L H ORCID: and Kamoun, Sophien ORCID: (2017) Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana. In: Wheat Rust Diseases. Methods in Molecular Biology . Humana Press, pp. 85-98. ISBN 978-1-4939-7248-7

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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.

Item Type: Book Section
Faculty \ School: Faculty of Science > The Sainsbury Laboratory
Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Plant Sciences
Depositing User: LivePure Connector
Date Deposited: 23 Jul 2018 10:30
Last Modified: 22 Oct 2022 00:07
DOI: 10.1007/978-1-4939-7249-4_8

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