Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.
Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.
Walter
Michael
M
Institut für Botanik und Botanischer Garten, Molekulare Entwicklungsbiologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany.
Chaban
Christina
C
Schütze
Katia
K
Batistic
Oliver
O
Weckermann
Katrin
K
Näke
Christian
C
Blazevic
Dragica
D
Grefen
Christopher
C
Schumacher
Karin
K
Oecking
Claudia
C
Harter
Klaus
K
Kudla
Jörg
J
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D008164:Luminescent Proteins; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014026:Nicotiana / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C104920:LSD1 protein, Arabidopsis; D008164:Luminescent Proteins; D010940:Plant Proteins; D014157:Transcription Factors; C495668:bZIP63 protein, Arabidopsis; C054562:yellow fluorescent protein, Bacteria