Protein–Protein Interaction Assays with Effector–GFP Fusions in Nicotiana benthamiana
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 wordsAgroinfiltration Live-cell imaging Affinity chromatography DNA assembly Proteomics
Research at The Sainsbury Laboratory (TSL) is supported by the Gatsby Charitable Foundation, the European Research Council (ERC), and the Biotechnology and Biological Sciences Research Council (BBSRC). We thank the Kamoun, Menke (proteomics), and the synthetic biology (Synbio) groups at TSL for continuous support and fruitful discussions. We also thank Dr. Thysis Buck for insightful discussions on protein–protein interaction assays.
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