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BREAKTHROUGH TECHNOLOGIES

Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants^{1,[C],[W],[OA]}

School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 20040, China (H.C., R.C.); National Institute of Biological Sciences, Beijing 102206, China (H.C., Y.Z., Y.S., H.L., Y.W., J.-M.Z.); and Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506 (X.T.)

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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jian-Min Zhou (zhoujianmin{at}nibs.ac.cn).

^[C] Some figures in this article are displayed in color online but in black and white in print.

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.111740

^* Corresponding author; e-mail zhoujianmin{at}nibs.ac.cn.

Received October 25, 2007; accepted November 30, 2007; published December 7, 2007.

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