title: A new β-estradiol-inducible vector set that facilitates easy construction and efficient expression of transgenes reveals CBL3-dependent cytoplasm to tonoplast translocation of CIPK5.
abstract: Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock (out/down) approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing β-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and StrepII epitope tags and harbor an optimized multiple cloning site for flexible and simple cloning strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL (Calcineurin B-like) protein expression on the subcellular localization of CIPKs (Calcineurin B-like interacting protein kinases). These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.
authors.affiliation: Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schlücking
authors.forename: Kai H
authors.initials: KH
authors.lastname: Edel
authors.forename: Philipp
authors.initials: P
authors.lastname: Köster
authors.forename: Maria M
authors.initials: MM
authors.lastname: Drerup
authors.forename: Christian
authors.initials: C
authors.lastname: Eckert
authors.forename: Leonie
authors.initials: L
authors.lastname: Steinhorst
authors.forename: Rainer
authors.initials: R
authors.lastname: Waadt
authors.forename: Oliver
authors.initials: O
authors.lastname: Batistic
authors.forename: Jörg
authors.initials: J
authors.lastname: Kudla
mesh_terms: D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004958:Estradiol / Q000737:chemistry*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D015388:Organelles / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D014026:Nicotiana / Q000235:genetics; D019076:Transgenes
chemical_list: D029681:Arabidopsis Proteins; C484079:CBL3 protein, Arabidopsis; D002135:Calcium-Binding Proteins; D049452:Green Fluorescent Proteins; D004958:Estradiol; D011494:Protein Kinases
keywords: Arabidopsis; CBL; CIPK.; inducible gene expression; vector; β-estradiol