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Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta
Authors:Citovsky Vitaly  Lee Lan-Ying  Vyas Shachi  Glick Efrat  Chen Min-Huei  Vainstein Alexander  Gafni Yedidya  Gelvin Stanton B  Tzfira Tzvi
Institution:Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Abstract: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.
Keywords:BiFC  bimolecular fluorescence complementation  EYFP  YFP variant derived from EGFP  nEYFP  N-terminal part of EYFP  cEYFP  C-terminal part of EYFP  YFP  yellow spectral variant  nYFP  N-terminal YFP  cYFP  C-terminal YFP  GFP  green fluorescent protein  ECFP  enhanced cyan variant of GFP  EGFP  enhanced GFP  MCS  multi-cloning site  MP  movement protein  pSATN  modular satellite plasmids  CaMV  cauliflower mosaic virus  ORF  open reading frame  NLS  nuclear localization signal  CRT  calreticulin  ChrD  cucumber chromoplast D protein  TYLCV  tomato yellow leaf curl virus  DIC  differential interference contrast  CP  capsid protein
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