Inoculation of Tobacco Protoplasts with Tobacco Rattle Virus: Experiments with Aggregates Formed in Inocula

Abstract

Summary Inocula made by mixing tobacco rattle virus (strain CAM; TRV), poly-l-ornithine (PLO) and either citrate buffer or phosphate buffer were compared by measuring their turbidity, by estimating the effect of centrifugation on their infectivity and by electron microscopy. Aggregates (floccules) containing virus particles were formed in the inocula but there was no evidence of any major difference in size between aggregates formed in the different buffers. The relationship between the loss of infectivity following centrifugation and the centrifugal force applied suggested that the aggregates causing infection sedimented at an average of 1340S (mol. wt. 7 × 108). Electron microscopy showed electron dense masses in most of which 10 to 20 TRV particles were embedded. Aggregates made in citrate had virus particles arranged radially whereas aggregates made in phosphate were more randomly constructed. Preparations of sedimented aggregates, and the supernatant fraction remaining following centrifugation were poorly infective unless PLO was added. In contrast with the addition of PLO to TRV preparations, adding PLO to these centrifuged fractions restored infectivity immediately. It is suggested that PLO can have two modes of action on infection when it is added to TRV to make inocula, one involving the formation of aggregates and the other for which aggregate formation is not essential.