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 language | English |
|---|---|
| Title of host publication | Wheat Rust Diseases |
| Subtitle of host publication | Methods and Protocols |
| Editors | Sambasivam Periyannan |
| Publisher | Humana Press |
| Chapter | 8 |
| Pages | 85-98 |
| Number of pages | 14 |
| ISBN (Electronic) | 978-1-4939-7249-4 |
| ISBN (Print) | 978-1-4939-7248-7 |
| DOIs | |
| Publication status | Published - 2017 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Publisher | Humana Press |
| Volume | 1659 |
| ISSN (Print) | 1064-3745 |
Profiles
-
Sophien Kamoun
- School of Biological Sciences - Professor of Biology
- Plant Sciences - Member
Person: Research Group Member, Academic, Teaching & Research
-
Frank Menke
- School of Biological Sciences - Honorary Associate Professor
- The Sainsbury Laboratory - Head of Proteomics (TSL)
Person: Honorary, Research & Analogous