Transient Overexpression of E2Fb to Induce Cell Divisions in Nicotiana benthamiana Pavement Cells

The availability of a fast and controlled mitotic model system that could simplify the generation of genetic material and reduce the experimental time from months to days would largely benefit research in plant cell division. In this protocol, we propose the use of pavement cells of Nicotiana benthamiana leaves to study cell division, which is artificially induced by Agrobacterium-mediated transient overexpression of the transcription factor E2Fb. The cell division-inducing overexpression of E2Fb can be combined with the expression of fluorescent protein-tagged proteins of interest or with dyes, which could be visualized throughout the cell cycle under the microscope. This simple and affordable method enables the study of cell cycle regulation and cell division in plants, from genome replication to cell wall formation, in a fast and controlled manner, and can be used for functional studies when coupled with chemical inhibitors or reverse genetic approaches.