title: Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.
abstract: Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.
authors.affiliation: Department of Evolutionary Biology and Biodiversity, National Institute for Basic Biology, Okazaki, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Kamigaki
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Nito
authors.affiliation: Department of Evolutionary Biology and Biodiversity, National Institute for Basic Biology, Okazaki, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hikino
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto, Japan.
authors.forename: Shino
authors.initials: S
authors.lastname: Goto-Yamada
authors.affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Nishimura
authors.affiliation: Department of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Organization for Research, Shimane University, Matsue, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Department of Evolutionary Biology and Biodiversity, National Institute for Basic Biology, Okazaki, Japan.|Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Mano
mesh_terms: D060054:Agrobacterium / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D059828:Plant Cells / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D060066:Protein Interaction Maps; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; D000097573:Red Fluorescent Protein; C527701:enhanced cyan fluorescent protein; C485184:enhanced green fluorescent protein