Time Course Analysis of Immunolocalization of Coat Protein and Replicase Proteins in Protoplasts Inoculated with Tobacco Mosaic Virus.

The localization of coat protein and putative viral replicase proteins (130 kDa and 180 kDa) of tobacco mosaic virus (TMV) in tobacco protoplasts was examined at different post-inoculation intervals, using specific antibodies and protein A-gold complex. Gold labels specific to antiserum against virus particle were first detected 4 hr postinoculation (p. i.) in small areas of the cytoplasm, where virus particles were not initially observed. At 6-15hr p.i. when virus multiplication was active, virus particles were accumulated and formed a paracrystalline array in the cytoplasm. Specific labels were found only on the crystalline aggregates and in the cytoplasm near the aggregates. Many virus aggregates were observed in each section of protoplasts at this stage. At 24 hr p.i., larger aggregates of virus particles were heavily labelled, while areas of the cytoplasm, where no virus particles were evident, were labelled scarcely. Nuclei, plastids and mitochondria were free of labels throughout the infection stages. Gold labels for 130 kDa protein were first detected on small TMV-specific inclusions in the cytoplasm at 4 hr p.i.: Thereafter, these inclusions grew both in number and size, and occurred frequently in the vicinity of virus aggregates. Labels were localized on the inclusions but not on virus aggregates throughout the infection stages. Those inclusions have dense labels and grew a little further in size, though virus replication at this stage declined to a low level. Gold labels for 180 kDa protein on the inclusions and other parts of protoplasts at each stage were observed similarly as the labels for 130 kDa protein. These observations indicate that replication of genomic RNA, synthesis of coat protein and subsequent virus assembly do take place at limited areas in the cytoplasm, where virus particles appeared and formed crystalline aggregate at later stages.