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

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationWheat Rust Diseases
Subtitle of host publicationMethods and Protocols
EditorsSambasivam Periyannan
PublisherHumana Press
Chapter8
Pages85-98
Number of pages14
ISBN (Electronic)978-1-4939-7249-4
ISBN (Print)978-1-4939-7248-7
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1659
ISSN (Print)1064-3745

Cite this