Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells

• Published in: The New phytologist 2011-04, Vol.190 (1), p.258-267
• Main Authors: Buschmann, H., Green, P., Sambade, A., Doonan, J. H., Lloyd, C. W.
• Format: Article
• Language: English
• Subjects: Actin; Actins; Agrobacterium; Agrobacterium - metabolism; Cell cycle; Cell division; Cells; Coculture Techniques; Cytokinesis; Cytoskeleton; Cytoskeleton - metabolism; Fluorescence; Fluorescence microscopy; Gene expression; Genetic transformation; HeLa cells; High frequency; In vivo methods and tests; Interphase; kinesin; Kinesins - chemistry; Kinesins - metabolism; Labeling; Labels; Localization; Metaphase; Methods; Microscopy; microtubule dynamics; Microtubules; Mitosis; Nicotiana - cytology; Plant cells; Plant Cells - metabolism; Plants; Plasmids - metabolism; Protein Structure, Tertiary; Tobacco; tobacco BY‐2; Transformation, Genetic; Transformations; transient gene expression; Western blotting
• ISSN: 0028-646X; 1469-8137
• DOI: 10.1111/j.1469-8137.2010.03587.x

• 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.