Abstract
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.
Highlights
The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions
bimolecular fluorescence complementation (BiFC) is relatively simple compared to fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) and has been used in a number of plant protein-protein interaction studies (Bracha-Drori et al, 2004; Walter et al, 2004; Dong et al, 2006; Quan et al, 2007)
We previously showed that the Pseudomonas syringae effector protein AvrB targets Arabidopsis (Arabidopsis thaliana) protein RAR1 to promote virulence (Shang et al, 2006)
Summary
The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The development of reporter-based in vivo proteinprotein interaction assays, such as fluorescence resonance energy transfer (FRET; Ha et al, 1996; Heim and Tsien, 1996; Mahajan et al, 1998), the related technology bioluminescence resonance energy transfer (BRET; Xu et al, 1999; Subramanian et al, 2006), and bimolecular fluorescence complementation (BiFC; Hu et al, 2002) assays, has significantly advanced the measurement of protein-protein interactions in vivo These assays are instrumental for a number of important discoveries in mammalian studies. Protein fragment complementation based on the reconstitution of murine dihydrofolate reductase (Remy and Michnick, 1999) was used to detect NPR1-TGA2 interaction in plants (Subramaniam et al, 2001) These assays typically require the addition of fluorescence-generating substrates and suffer from the pitfalls of FRET and BiFC. The utility of the firefly LCI in plant protein-protein interaction studies remains to be tested
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