A Cell-Free Replication System for Positive-Strand RNA Viruses for Identification and Characterization of Plant Resistance Gene Products

: Plant cells have lytic vacuoles, which contain ribonucleases and proteinases. The vacuoles are fragile and easily collapsed upon homogenization of plant tissues or cells. Thus, with a few exceptions, plant cell extracts are contaminated by vacuole-derived lytic enzymes and unsuitable for biochemical analyses. Here, we describe a method for removing the vacuoles from intact tobacco BY-2 protoplasts and for cell-free translation and replication of genomic RNA of positive-strand RNA viruses using the extract of evacuolated protoplasts. We also describe a method for the identification and functional characterization of a plant resistance gene product using this system.