Plant Science Innovation, Center for
A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice
Date of this Version
MOLECULAR PLANT PATHOLOGY (2006) 7(5), 417–427
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.
© 2006 BLACKWELL PUBLISHING LTD