Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.
Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.
Citovsky
Vitaly
V
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Lee
Lan-Ying
LY
Vyas
Shachi
S
Glick
Efrat
E
Chen
Min-Huei
MH
Vainstein
Alexander
A
Gafni
Yedidya
Y
Gelvin
Stanton B
SB
Tzfira
Tzvi
T
D001681:Biological Assay; D015415:Biomarkers / Q000378:metabolism; D002451:Cell Compartmentation; D019281:Dimerization; D018274:Electroporation; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D059828:Plant Cells; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins