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

Songbiao Chen, Lizen Tao, Lirong Zeng, Miguel E. Vega-Sanchez, Kenji Umemura, Guo Liang Wang

Research output: Contribution to journalArticle

235 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)417-427
Number of pages11
JournalMolecular Plant Pathology
Volume7
Issue number5
DOIs
StatePublished - Sep 2006

ASJC Scopus subject areas

  • Molecular Biology
  • Agronomy and Crop Science
  • Soil Science
  • Plant Science

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