Journal Article

Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants

Lin, Huiqiong, Wang, Yujing, Zou, Yan, Zhou, Jian-Min, Tang, Xiaoyan, Shang, Yulei, Chen, Huamin, Cai, Run

2008

Overview

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

• Publisher: American Society of Plant Biologists,American Society of Plant Physiologists
• Subject: Arabidopsis / Arabidopsis - genetics / Arabidopsis - metabolism / bacterial proteins / Biological and medical sciences / Breakthrough Technologies / Cell biochemistry / Cell physiology / Complementation / disease resistance / energy transfer / Fluorescence / Fundamental and applied biological sciences. Psychology / Gene Expression Regulation, Plant / Genetic Complementation Test / Genetic Complementation Test - methods / Genetic Variation / genetics / HSP90 Heat-Shock Proteins / HSP90 Heat-Shock Proteins - genetics / HSP90 Heat-Shock Proteins - metabolism / image analysis / Imaging / instrumentation / Leaves / luciferase / Luciferases, Firefly / Luciferases, Firefly - metabolism / metabolism / methods / mutants / Nicotiana - genetics / Nicotiana - metabolism / Plant cells / Plant interaction / Plant physiology and development / plant proteins / Plant Proteins - genetics / Plant Proteins - metabolism / Plants / Protein Binding / protein synthesis / protein-protein interactions / Proteins / Protoplasts / Pseudomonas syringae / Rhizobium / SKP Cullin F-Box Protein Ligases / SKP Cullin F-Box Protein Ligases - genetics / SKP Cullin F-Box Protein Ligases - metabolism / Tobacco / transcription factors / Transcription Factors - genetics / Transcription Factors - metabolism / transgenic plants / Yeasts