Infection of Tobacco Mesophyll Protoplasts with Raspberry Ringspot Virus alone and together with Tobacco Rattle Virus

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Abstract
Raspberry ringspot virus (RRV) infected tobacco mesophyll protoplasts best, judged by the yield of infective virus, when the inoculation mixture contained 0.04-5 .mu.g/ml virus, 1.0-1.5 .mu.g/ml poly-L-ornithine and phosphate buffer (0.006-0.025 M, pH 6.0-9.0); without poly-L-ornithine no infection occurred. The optimum temperature for accumulation of infective virus was 20-22.degree. C, and the virus content reached about 2 .times. 106 particles/protoplast in 3 days at 22.degree. C. On staining with fluorescent antibody to purified virus particles, infected protoplasts gave a faint generalized cytoplasmic fluorescence, with many also containing a more brightly fluorescing spot thought to correspond with the vesiculated inclusion body found by EM. In contrast, many of the protoplasts inoculated with mixtures of RRV and the CAM strain of tobacco rattle virus (TRV-CAM) contained numerous discrete granules of RRV particle antigen throughout the cytoplasm. Infection with RRV had little effect on the distribution of TRV-CAM particle antigen or on the proportion of protoplasts infected with TRV-CAM. Up to 95% of protoplasts became infected with both viruses, which apparently infected independently. The aggregates of RRV antigen formed only in protoplasts also infected with TRV-CAM.