The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants

Abstract

The 2 M LiCl-soluble RNA fraction extracted from tobacco mosaic virus (TMV)-infected tobacco plants contains, in addition to the viral replicative form of 4 × 10 6 MW, three smaller double-stranded (ds) RNA species with apparent molecular weights (estimated by polyacrylamide gel electrophoresis, using ds RNAs as markers) of 2.25, 1.1, and 0.23 × 10 6. The synthesis of all four ds RNAs is insensitive to actinomycin D. They are completely RNase insensitive at high salt concentrations and are found both in directly inoculated and in apical tissues. In tissues incubated in the presence of 3H-uridine and actinomycin D, the three small ds RNAs accounted for 6 to 11.5% of the total radioactivity incorporated into viral ds RNA. On a molar basis, however, in apical leaves the smallest ds RNA was synthesized to almost the same level as the replicative form. By molecular hybridization, the three small ds RNAs have been shown to be of viral origin, and each contains sequences represented in the 5′ end of complementary (negative strand) TMV RNA. Based on molecular weight data, none of the ds RNAs can be considered to be a ds form of the subgenomic TMV coat protein mRNA (the LMC), suggesting that it is not replicated independently. None of the small ds RNAs was found to be an endogenous product of the bound TMV RNA replicase.