Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta |
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Authors: | Citovsky Vitaly Lee Lan-Ying Vyas Shachi Glick Efrat Chen Min-Huei Vainstein Alexander Gafni Yedidya Gelvin Stanton B Tzfira Tzvi |
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Affiliation: | Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA. |
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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. |
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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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