Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants1[C][W][OA]
By Huamin Chen, Yan Zou, Yulei Shang, Huiqiong Lin, Yujing Wang, R. Cai, Xiaoyan Tang, Jian-Min Zhou. Plant Physiology, 2007
1,017 citations recorded by the source corpus.
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.
Subfields: Insect Science, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/8ecc78a29d7ee0a17c5700e90b840bcbd8cf301e
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Citations in the corpus
- A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes (2011, methodology): “complementation (BiFC) [30] and firefly luciferase complementation (FLC) assays [31].”
- Negative Regulation of Anthocyanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor[W][OA] (2011, methodology): “LUC activity was barely detectable in leaves infiltrated with combinations of Pro35S:nLUC and Pro35S:cLUC, Pro35S:cLUC and Pro35S:rSPL9-nLUC, or Pro35S:nLUC and Pro35S:cLUC-PAP1.”
- Gateway Vectors for Plant Genetic Engineering: Overview of Plant Vectors, Application for Bimolecular Fluorescence Complementation (BiFC) and Multigene Construction (2012, neutral): “, 2004), BRET(Chen et al., 2008; Xu et al., 2007), fluorescence”