A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice

Authors

• Songbiao Chen, The Ohio State University
• Lizen Tao, South China Agricultural University
• Lirong Zeng, The Ohio State University
• Miguel E. Vega-Zanchez, The Ohio State University
• Kenji Umemura, Meiji Seika Kaisha Ltd
• Guo-Liang Wang, The Ohio State UniversityFollow

Document Type

Article

Date of this Version

2006

Citation

MOLECULAR PLANT PATHOLOGY (2006) 7(5), 417–427

Comments

© 2006 BLACKWELL PUBLISHING LTD

DOI: 10.1111/J.1364-3703.2006.00346.X

Abstract

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.