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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References
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Ishibashi, K., Ishikawa, M. (2019). A Cell-Free Replication System for Positive-Strand RNA Viruses for Identification and Characterization of Plant Resistance Gene Products. In: Kobayashi, K., Nishiguchi, M. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 2028. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9635-3_6
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DOI: https://doi.org/10.1007/978-1-4939-9635-3_6
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Publisher Name: Humana, New York, NY
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