Entry of Tobacco Mosaic Virus RNA into Tobacco Chloroplasts in Vivo and in Vitro

Abstract

Chloroplasts may be adversely affected in tobacco mosaic virus (TMV)-infected plants. Part of the perturbation could result from the known accumulation of virus and viral-coded components within that organelle. Our studies have been directed at ascertaining how these virus-related components gain entry into the chloroplasts. We prepared chloroplasts from either directly-inoculated or systemically-infected leaves of TMV-infected tobacco, and from uninfected controls. Intact chloroplasts, isolated from Percoll gradients, were treated with both pancreatic ribonuclease and thermolysin to destroy any TMV particles and RNA, either bound to or copurifying with the chloroplasts. RNA isolated from such chloroplasts was probed on Northern blots with ³²P-labelled cDNA prepared from TMV RNA. Genomic, but not sub-genomic TMV RNA was detected within the chloroplasts of directly-inoculated and systemically-invaded leaves, indicating that the uptake into the chloroplast is selective. Evidence showing that TMV RNA per se (as opposed to virions) could enter is based on the observation that the temperature-sensitive mutant Ts 38, in which no virions are formed at a restrictive temperature of 35°, still allows for entry of the viral RNA at that temperature.

An in vitro system has been developed in which isolated chloroplasts are incubated with labelled TMV RNA, or other RNAs as controls. As in the leaf, the uptake is specific to the genomic length TMV RNA, with subgenomic and non-viral RNAs being excluded. The parameters of this system are being optimized, and the sequences of genomic TMV RNA essential for uptake are being determined.