Transient transformation and RNA silencing in Zinnia tracheary element differentiating cell cultures
The Zinnia elegans cell culture system is a robust and physiologically relevant in vitro system for the study of xylem formation. Freshly isolated mesophyll cells of Zinnia can be hormonally induced to semisynchronously transdifferentiate into tracheary elements (TEs). Although the system has proven to be valuable, its utility is diminished by the lack of an efficient transformation protocol. We herein present a novel method to introduce DNA/RNA efficiently into Zinnia cells by electroporation-based transient transformation. Using reporter gene plasmids, we optimized the system for efficiency of transformation and ability for the transformed cells to transdifferentiate into TEs. Optimal conditions included a partial digestion of the cell walls by pectolyase, a low voltage and high capacitance electrical pulse and an optimal medium to maintain cell viability during transformation. Beyond the simple expression of a reporter protein in Zinnia cells, we extended our protocol to subcellular protein targeting, simultaneous co-expression of several reporter proteins and promoter-activity monitoring during TE differentiation. Most importantly, we tested the system for double-stranded RNA (dsRNA)-induced RNA silencing. By introducing in vitro-synthesized dsRNAs, we were able to phenocopy the Arabidopsis cellulose synthase (CesA) mutants that had defects in secondary cell-wall synthesis. Suppressing the expression ofZinnia CesA homologues resulted in an increase of abnormal TEs with aberrant secondary walls. Our electroporation-based transient transformation protocol provides the suite of tools long required for functional analysis and developmental studies at single cell levels.
Document Type: Research Article
Affiliations: 1: RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045 Japan 2: UMR-CNRS-UPS 5546 «Surfaces Cellulaires et Signalisation chez les Vegetaux», Pole de Biotechnologie Vegetale, 24 Chemin de Borde-Rouge, 31326 Castanet-Tolosan, France 3: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan
Publication date: March 1, 2008