Infection of Tobacco Mesophyll Protoplasts with Tobacco Necrotic Dwarf Virus, a Phloem-limited Virus

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Abstract
Tobacco mesophyll protoplasts were successfully inoculated with tobacco necrotic dwarf virus (TNDV), a virus closely related to potato leafroll virus. The optimum conditions for infection as assessed by staining with fluorescent antibody to virus particles were 1 .mu.g/ml TNDV, 1 .mu.g/ml poly-L-ornithine, 0.025 M phosphate buffer, pH 5.4 and 1-2 .times. 105/ml protoplasts; this combination resulted in 70-80% infection. Accumulation of virus antigen was first detected 12 h after inoculation; the number of fluorescing protoplasts and intensity of fluorescence increased markedly during the following 20 h. Virus antigen was distributed throughout the cytoplasm. EM of thin sections revealed groups of virus-like particles in the cytoplasm and a few similar particles in the nucleus. Virus yield was estimated to be 7.8 .times. 105 particles/infected protoplast at 30 h after inoculation. Accumulation of virus antigen was completely inhibited by cycloheximide but was not affected by actinomycin D or chloramphenicol when they were applied 2 h after inoculation. Studies on the replication of aphid-borne, phloem-limited viruses should be greatly facilitated by using the mesophyll protoplast system.