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Uptake of Tobacco Rattle Virus by Tobacco Protoplasts, and the Effect of Phosphate on Infection
Abstract
SUMMARY
In the presence of poly-l-ornithine at 1 µg/ml, the uptake of [3H]-labelled particles of tobacco rattle virus (TRV) by tobacco mesophyll protoplasts during inoculation depended on the virus and protoplast concentrations in the inoculation mixture. Uptake decreased 15- to 30-fold in the absence of poly-l-ornithine. Substituting phosphate for citrate buffer in the inoculum greatly increased infection but had little effect on virus uptake. For infections detected by staining with fluorescent antibody to virus particles, the ID50 occurred at an uptake of 30 long TRV particles per protoplast using phosphate buffer, and 250 using citrate. Evidence was obtained that the phosphate-induced enhancement of infection is mediated through an effect on interaction of virus and poly-l-ornithine during pre-inoculation incubation. Batches of protoplasts differing in susceptibility to infection did not differ similarly in uptake of inoculum virus.
Electron microscopy of sections of freshly inoculated protoplasts revealed single TRV particles associated end-on with normal-looking plasmalemma, and aggregates that contained densely stained material plus TRV particles, associated with breaks in the plasmalemma, and adjacent to areas of intracytoplasmic vesiculation. Some aggregates entered the cytoplasm and some TRV particles entered the vesicles. The aggregates were on average smaller after inoculation with phosphate-containing than with citrate-containing inocula. It is suggested that mini-aggregates of the kind produced during incubation with phosphate play an important role in infection.
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© Journal of General Virology 1976