A Cell-Free Replication System For Positive-Strand Rna Viruses For Identification And Characterization Of Plant Resistance Gene Products
ANTIVIRAL RESISTANCE IN PLANTS: METHODS AND PROTOCOLS(2019)
Abstract
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.
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Key words
BYL, Tomato mosaic virus, Tm-1, Translation, Replication
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