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Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium ‐mediated transient transformation of tobacco BY‐2 cells
Author(s) -
Buschmann H.,
Green P.,
Sambade A.,
Doonan J. H.,
Lloyd C. W.
Publication year - 2011
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/j.1469-8137.2010.03587.x
Subject(s) - phragmoplast , mitosis , interphase , cytokinesis , cell plate , microbiology and biotechnology , agrobacterium , biology , telophase , cell division , transformation (genetics) , cytoskeleton , cell cycle , microtubule , metaphase , anaphase , cell , genetics , gene , chromosome
Summary• 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.