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

Abstract

SUMMARY 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 µg/ml TNDV, 1 µg/ml poly-l-ornithine, 0.025 m-phosphate buffer, pH 5.4, and 1 to 2 × 105/ml protoplasts: this combination resulted in 70 to 80% infection. Accumulation of virus antigen was first detected 12 h after inoculation, and both the number of fluorescing protoplasts and intensity of fluorescence increased markedly during the following 20 h. Virus antigen was distributed throughout the cytoplasm. Electron microscopy 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 × 105 particles per 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 aphidborne, phloem-limited viruses should be greatly facilitated by using the mesophyll protoplast system.