Inoculation of cowpea mesophyll protoplasts with unseparated RNA 1 and RNA 2 from red clover necrotic mosaic virus in the presence of polyethylene glycol resulted in virus RNA replication, the synthesis of virus capsid polypeptide and the formation of virus particles; 75 to 85% of the viable protoplasts became infected and the yield of virus particles or virus RNA after 72 h incubation corresponded to about 3 × 10 virus genomes per infected protoplast. In contrast, no replication could be detected when protoplasts were inoculated with RNA 2 alone. However, inoculation of protoplasts with RNA 1 alone resulted in its replication and the formation of virus particles, with a yield similar to that obtained after inoculation with both RNAs. Since infection of plants requires both RNA 1 and RNA 2 to be present, the demonstration of the independent replication of RNA 1 in single cells strengthens the hypothesis that RNA 2 plays a role in the cell-to-cell transmission of the virus.


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