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Published on December 7, 2007; 10.1104/pp.107.111740

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Received October 25, 2007
Accepted November 30, 2007

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

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

School of Agriculture and Biology, Shanghai Jiaotong University; National Institute of Biological Sciences, Beijing; Department of Plant Pathology, Kansas State University

* Corresponding author; email: zhoujianmin{at}nibs.ac.cn.

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 (FRET) and Bimolecular Fluorescence Complementation (BiFC) that are used widely for protein-protein interaction studies in plants require extensive instrumentation. To facilitate protein-protein interactions studies in plants, we adopted the luciferase complementation imaging (LCI) 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 9 pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SCF 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.




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