High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions

• Published in: The Plant journal : for cell and molecular biology Vol. 41; no. 1; pp. 162 - 174
• Main Authors: Koroleva, O.A, Tomlinson, M.L, Leader, D, Shaw, P, Doonan, J.H
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-01-2005; Blackwell Science; Blackwell Publishing Ltd
• Subjects: Agrobacterium; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Arabidopsis; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biological and medical sciences; cell culture; cell cycle; cell suspension culture; Diverse techniques; DNA-binding proteins; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gene expression; gene expression regulation; gene fusion; Gene Transfer Techniques; genetic transformation; genetic vectors; Genomics; GFP fusion; green fluorescent protein; Green Fluorescent Proteins - genetics; high throughput; Molecular and cellular biology; Open Reading Frames; Plant populations; plant proteins; protein kinases; protein localization; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; reporter genes; subcellular compartments; transcription factors; transgenic plants; transient expression; Arabidopsis; cell cycle; subcellular compartments; Experimental protocol; high throughput; Protein; protein localization; Arabidopsis thaliana; transient expression; Gene; Cruciferae; cell culture; Dicotyledones; Angiospermae; Spermatophyta; Molecular cloning; Localization; GFP fusion
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2004.02281.x

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.