title: Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies.
abstract: Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.).
authors.forename: Andreas
authors.initials: A
authors.lastname: Hecker
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.).
authors.forename: Niklas
authors.initials: N
authors.lastname: Wallmeroth
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.).
authors.forename: Sébastien
authors.initials: S
authors.lastname: Peter
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.).
authors.forename: Michael R
authors.initials: MR
authors.lastname: Blatt
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.).
authors.forename: Klaus
authors.identifier: http://orcid.org/0000-0002-2150-6970
authors.initials: K
authors.lastname: Harter
authors.affiliation: University of Tübingen, Centre for Plant Molecular Biology-Plant Physiology (A.H., K.H.), Centre for Plant Molecular Biology-Developmental Genetics (N.W., C.G.), and Institut für Physikalische und Theoretische Chemie (S.P.), D-72076 Tuebingen, Germany; andUniversity of Glasgow, Laboratory of Plant Physiology and Biophysics, Glasgow G12 8QQ, United Kingdom (M.R.B.) christopher.grefen@uni-tuebingen.de.
authors.forename: Christopher
authors.identifier: http://orcid.org/0000-0002-5820-4466
authors.initials: C
authors.lastname: Grefen
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D038761:Photobleaching; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D010940:Plant Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins