Distribution of tobacco mosaic virus RNA molecules in infected tobacco protoplasts revealed by in situ hybridization

The distribution of virus RNA molecules in tobacco mosaic virus (TMV) infected tobacco protoplasts was examined with time after inoculation using in situ hybridization with strand specific riboprobe labeled with digoxigenin. Analysis of hybridization signals for TMV plus strand RNAs showed very weak signals throughout the cytoplasm of the protoplasts sampled at 2hr post-inoculation (p.i.). Thereafter, intensity of the signals increased in the cytoplasm and reached a maximum level at 12hr p.i. There was then a rapid decrease in the intensity of signals. Weak signals in the in situ hybridization using a minus strand specific probe also were first observed at 2hr p.i. The intensity of signals, then increased and gradually decreased after a maximum level at 12hr p.i. Intensity of the signals at a maximum level, however, was much less than that of plus strand viral RNA. No hybridization signals for plus strand RNAs or minus strand RNAs was demonstrated in the nucleus throughout the period of infection. These results strongly indicated that the site of replication of TMV-RNA is the cytoplasm and that the nucleus is not directly associated with its synthesis.