The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread. INTRODUCTIONMost plant viruses encode one or more proteins that are required to achieve local and systemic invasion of the host. These so-called movement proteins (MPs) enable viruses to exploit plasmodesmata, the gated, plasma membrane-lined channels that provide symplastic continuity between adjacent cells and through which plant cells communicate (Epel, 1994;Lucas and Gilbertson, 1994; Fenczik et al., 1995).Pioneering studies of MP functions were performed with the MP of tobacco mosaic virus (TMV) (Deom et al., 1987;Meshi et al., 1987). In infected tobacco plants as well as in transgenic plants, the MP accumulates in plasmodesmata and increases their size exclusion limit (Tomenius et al., 1987;Wolf et al., 1989; Atkins et al., 1991a; Ding et al., 1992;Moore et al., 1992;Oparka et al., 1997). The protein also binds single-stranded nucleic acids in vitro, resulting in unfolded and elongated protein-nucleic acid complexes.This observation led to the hypothesis that the virus moves from cell to cell in the form of a viral ribonucleoprotein complex (vRNP) that in size and structure is compatible with the modified plasmodesmata (Citovsky et al., 1990(Citovsky et al., , 1992.Although it is evident that the MP and vRNP must enlist cytoplasmic structures to aid transfer from their site of synthesis to the plasmodesmata, little is known about the nature of these components and about the targeting mechanism per se. F-actin and microtubules were proposed as targeting systems for MP (Heinlein et al., 1995;McLean et al., 1995; Carrington et al....

No abstract

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.