Studies on a Nicotiana hybrid infected with a plant rhabdovirus

The morphology of Sonchus yellow net virus (SYNV) has been studied by electron microscopy in both negative stained and ultrathin sectioned samples. Depending on the stain, bullet-shaped or bacilliform particles could be observed. However, the incubation of grids containing the samples with anti-SYNV antiserum prior to negative staining preserved the integral bacilliform morphology of the virus particles. The yield of the virus during the purification-steps was estimated by enzyme-linked immunosorbent assay (ELISA). The loss of the virus, using the standard procedure for purification was about 24%. Systemic movement of SYNV to leaves and roots was first detected by protein immunoblotting and ELISA 24 h after mechanical inoculation. Virus levels rose to a maximum ten days after inoculation: the highest levels, between 4.0 and 7.3 mug/g tissue, were in leaves which were not yet fully expanded. Electron microscopy of tissue sections revealed that when the virus content of tissues was greatest, virtually all leaf and root cells were infected. Most of the virions were in the perinuclear space, with only a few scattered particles in the cytoplasm. Nuclei contained large viroplasms associated with viral nucleocapsids, the matrix of these viroplasms reacted strongly to anti-SYNV antiserum in immunogold labelling experiments. Between 10 and 20 days after inoculation, levels of virus antigen and viral RNA fell to about 20% of their maximum. By 20 days after inoculation, no more than 10% of cells contained virus particles and almost all the virions were within the cytoplasm. Virions were almost never observed in most tissues of plants infected for longer than 60 days. These results suggest that SYNV spreads systemically until most or all cells are infected. The plants then undergo a recovery phase during which virions disappear from the nuclei of infected cells and vesiculate into the cytoplasm. The effects of SYNV on the nucleus, chloroplasts, mitochondria, endoplasmic reticulum, plasma membranes and the cell walls in mechanically inoculated N. edwardsonii were studied at various times after inoculation. Immunogold labelling was used to localize the viral protein(s) in infected cells. During the acute phase of the virus infection, nuclei showed obvious abnormalities, most strikingly, the development of nuclear viroplasms containing viral nucleocapsids and granular or fibrillar matrix. The association of the nucleocapsids with viroplasms and the strong reaction, in immunogold labelling experiments, of gold particles to these viroplasms suggest that viroplasms are the sites of nucleocapsid assembly. Chloroplasts of infected cells exhibited a number of ultrastructural abnormalities. In immunogold labelling experiments, antiserum to purified SYNV bound extensively to the thylakoids and stroma of chloroplasts from infected cells at all stages of infection, but not to the vesicles or inclusion bodies. Mitochondria of infected cells also exhibited a number of ultrastructural abnormalities. Neither nucleocapsids nor virus particles were observed in association with diseased mitochondria and in immunogold labelling experiments, no label was bound to mitochondria. Changes in the endoplasmic reticulum (ER) network were observed. Nucleocapsids and virus particle were associated with this ER. Infected cells showed alteration in the plasma membranes including the formation of piasmalemmasomes, multivesiculated plasmalemmasomes and piasmalemmasome-like structures. Tubular channels interconnecting adjacent cells and often containing virions developed. Similar channels containing nucleocapsids were observed within the nucleus and interconnecting the nucleus and the cell wall. Immunogold labelling indicated the presence of viral proteins associated with the cell wall or associated structures. These channels may be involved in movement of virus from cell to cell. Virus particles were not detectable by electron microscopy in chronically infected plants. However, virus proteins G & N plus a novel immunologically cross-reacting polypeptide of 41 KD (p41) were detectable in immunoblots. Immunogold labelling experiments revealed the presence of considerable quantities of free virus protein in the nucleus and cytoplasm. In leaf discs labelled with 35S-methionine, synthesis, in vivo, of all four virus structural proteins was detectable. Proteins N, M1 & M2 were detected in the in vitro translation products of poly(A)+mRNA from these plants. Thus proteins G & N accumulate in leaves but M1 & M2 fail to do so, presumably as a result of rapid turnover or specific degradation. The origin of protein p41 is not clear. It may be an additional non-structural viral protein, a modified form of one of the other structural proteins or a cross-reacting host protein induced by chronic infection. Plants were examined by electron microscopy 5 months after inoculation with SYNV. Mo virions were observed in leaf or root cells, but cells in sections of calyx contained large numbers of virus particles. Most particles were only 73-86% of the length of standard SYNV but reacted with anti-SYNV antiserum in immunogold labelling. Plants inoculated with sap extracted from calyx became systemically infected but exhibited chlorotic mottling, instead of the normal vein-clearing symptoms. Most virus particles in these plants were short, and when purified, sedimented more slowly than standard SYNV. Purified short particles were not infective, but plants inoculated with a mixture of short and standard particles developed mottling symptoms and yielded predominantly short particles. Proteins from short particles were electrophoretically and antigenically identical to those from standard virus. RNA from short particles was about 77% the size of RNA from standard SYNV and hybridized to cloned SYNV cDNA. These short particles have all the characteristics of defective-interfering particles. When plants were infected using inocula derived from chronically infected plants, nucleocapsids were observed within chloroplasts. Western blots of protein from chloroplasts isolated from these plants revealed the presence of the virus nucleocapsid protein N and possibly protein L