title: Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.
abstract: Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle.
authors.affiliation: Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
authors.forename: H
authors.initials: H
authors.lastname: Buschmann
authors.affiliation: Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
authors.forename: P
authors.initials: P
authors.lastname: Green
authors.affiliation: Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
authors.forename: A
authors.initials: A
authors.lastname: Sambade
authors.affiliation: Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
authors.forename: J H
authors.initials: JH
authors.lastname: Doonan
authors.affiliation: Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
authors.forename: C W
authors.initials: CW
authors.lastname: Lloyd
mesh_terms: D060054:Agrobacterium / Q000378:metabolism*; D018920:Coculture Techniques; D048749:Cytokinesis; D003599:Cytoskeleton / Q000378:metabolism*; D007399:Interphase; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism; D008938:Mitosis; D059828:Plant Cells / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014026:Nicotiana / Q000166:cytology*; D014170:Transformation, Genetic
chemical_list: D016547:Kinesins