Establishment of a mitotic model system by transient expression of the D-type cyclin in differentiated leaf cells of tobacco (Nicotiana benthamiana)

Abstract

Investigations of plant cell division would greatly benefit from a fast, inducible system. Therefore, we aimed to establish a mitotic model by transiently expressing D-type cyclins in tobacco leaf cells. Two different D-type cyclins, CYCD3;1 and CYCD4;2 from Arabidopsis thaliana, were expressed by agrobacterial infiltration in the cells of expanded leaves in tobacco (Nicotiana benthamiana). Leaf pavement cells were examined after cyclin expression while target and reference (histone or tubulin) proteins were marked by fluorescent protein-tagging. Ectopic expression of the D-type cyclin induced pavement cells to re-enter cell division by establishing mitotic microtubule arrays. The induced leaf cells expressed M phase-specific genes of Arabidopsis encoding the mitotic kinase AtAurora 1, the microtubule-associated proteins AtEDE1 and AtMAP65-4, and the vesicle fusion protein AtKNOLLE by recognizing their genomic elements. Their distinct localizations at spindle poles (AtAurora1), spindle microtubules (AtEDE1), phragmoplast microtubules (AtMAP65-4) and the cell plate (AtKNOLLE) were indistinguishable from those in their native Arabidopsis cells. The dividing cells also revealed two rice (Oryza sativa) microtubule-associated proteins in the phragmoplast and uncovered a novel spindle-associated microtubule motor protein. Hence, this cell division-enabled leaf system predicts hypothesized cell cycle-dependent functions of heterologous genes by reporting the dynamics of encoded proteins.

Keywords: cell division-enabled leaf system (CDELS); cyclin D; microtubules; mitosis; phragmoplast; spindle; tobacco; transient expression.