title: A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.
abstract: SUMMARY The transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stem- and sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate large-scale functional analysis of defence-related genes in rice.
authors.affiliation: Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Songbiao
authors.initials: S
authors.lastname: Chen
authors.forename: Lizen
authors.initials: L
authors.lastname: Tao
authors.forename: Lirong
authors.initials: L
authors.lastname: Zeng
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Vega-Sanchez
authors.forename: Kenji
authors.initials: K
authors.lastname: Umemura
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Wang