Firefly luciferase complementation imaging assay for protein-protein interactions in plants.
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.
Chen
Huamin
H
School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 20040, China.
Zou
Yan
Y
Shang
Yulei
Y
Lin
Huiqiong
H
Wang
Yujing
Y
Cai
Run
R
Tang
Xiaoyan
X
Zhou
Jian-Min
JM
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test / Q000379:methods*; D014644:Genetic Variation; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D049409:Luciferases, Firefly / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012231:Rhizobium; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D010940:Plant Proteins; D014157:Transcription Factors; D049409:Luciferases, Firefly; D044843:SKP Cullin F-Box Protein Ligases