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.

A method for studying cell division in Nicotiana benthamiana pavement cells based on Agrobacterium infiltration

“…This showed that the CDELS method can provide fast localization data for novel cell division genes based on live‐cell microscopy. Taken together, the paper by Xu et al . provides a rapid method to analyse cell division genes upon Agrobacterium infiltration of N. benthamiana leaves.…”

“…Now, in this issue of New Phytologist , Xu et al (2020; pp. 1213–1221) present a simple modification of the N. benthamiana – Agrobacterium infiltration method (Sparkes et al , 2006) to induce cell division in leaf epidermal pavement cells.…”

“…Via downstream signalling CDK‐mediated phosphorylation results in the activation of transcription factor E2F to induce cell cycle progression (Polyn et al , 2015). Consequently, Xu et al . employed CYCD genes from Arabidopsis (CYCD4;2 and CYCD3;1), which upon infiltration and transient overexpression (via the CaMV35S promoter) pushed the differentiated pavement cells back into the cell cycle.…”

“…Xu et al next analysed known cell division genes using CDELS and compared the observed protein localization with previously published results. For this they chose AURORA , EDE1 , MAP65 and KNOLLE genes from Arabidopsis , fused them to GFP sequences and infiltrated the fusions alongside CYCD into tobacco leaves.…”

“…This is simply fascinating as the method is apparently easy to handle and certainly available to most laboratories studying plants. The publication by Xu et al . showcases FP‐tagged markers (plant genes of interest) that are expressed from their native promoters.…”