Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection.

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.     

Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.     

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.     

Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection

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.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.     

Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

绿色荧光蛋白标记检测鸦胆子素D抗TMV活性

以绿色荧光蛋白（green fluorescent protein, GFP）为报告基因,将含TMV表达载体的质粒p35S-30B：GFP转化农杆菌EHA 105,通过渗透法把经MMA诱导后的农杆菌悬浮液注射到本氏烟叶片内,测定了鸦胆子素D （Bruceine D） 对烟草植株内TMV的增殖和运动的抑制作用;通过PEG介导法把p35S-30B：GFP转化到本氏烟叶肉细胞原生质体内,测定了Bruceine D对烟草原生质体中TMV增殖的抑制效果.结果表明,在10 μg/mL浓度下,Bruceine D不仅可抑制烟草叶肉细胞原生质体中TMV的增殖,还可以抑制烟草接种叶中TMV向茎部及植株上部叶片移动,且对寄主植物不造成明显的毒害.     

Identification of Ourmiavirus 30K movement protein amino acid residues involved in symptomatology,viral movement,subcellular localization and tubule formation

Several plant viruses encode movement proteins (MPs) classified in the 30K superfamily. Despite a great functional diversity, alignment analysis of MP sequences belonging to the 30K superfamily revealed the presence of a central core region, including amino acids potentially critical for MP structure and functionality. We performed alanine‐scanning mutagenesis of the Ourmia melon virus (OuMV) MP, and studied the effects of amino acid substitutions on MP properties and virus infection. We identified five OuMV mutants that were impaired in systemic infection in Nicotiana benthamiana and Arabidopsis thaliana, and two mutants showing necrosis and pronounced mosaic symptoms, respectively, in N. benthamiana. Green fluorescent protein fusion constructs (GFP:MP) of movement‐defective MP alleles failed to localize in distinct foci at the cell wall, whereas a GFP fusion with wild‐type MP (GFP:MPwt) mainly co‐localized with plasmodesmata and accumulated at the periphery of epidermal cells. The movement‐defective mutants also failed to produce tubular protrusions in protoplasts isolated from infected leaves, suggesting a link between tubule formation and the ability of OuMV to move. In addition to providing data to support the importance of specific amino acids for OuMV MP functionality, we predict that these conserved residues might be critical for the correct folding and/or function of the MP of other viral species in the 30K superfamily.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Degradation of tobacco mosaic virus movement protein by the 26S proteasome

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and clasto-lactacystin-beta-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.     

Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.     

Odontoglossum ringspot virus host range restriction in Nicotiana sylvestris maps to the replicase gene

The experimental host range of Odontoglossum ringspot virus (ORSV), a member of the tobamoviruses, includes several species of Nicotiana , but not N. sylvestris . However, ORSV was able to replicate in protoplasts from N. sylvestris leaves. By using the green fluorescent protein (GFP) as a marker inserted into ORSV, it was found that a small number of single epidermal cells became infected in mechanically inoculated leaves, but the virus did not move cell to cell. The ORSV movement protein (MP) and coat protein (CP) were examined for their ability to effect movement by substitution into Tobacco mosaic virus (TMV) hybrids. Both proteins and the 3' non-translated region (NTR) of ORSV allowed movement of TMV hybrids in N. sylvestris . These results suggested that the inability of ORSV to move in N. sylvestris was due to the replicase gene or the 5'NTR. One possibility was that the replicase gene could indirectly affect movement by failing to produce subgenomic (sg) RNAs for expression of MP or CP, but this appeared not to be the case as ORSV replicated and produced MP and CP sgRNAs, both of which were translated in N. sylvestris protoplasts. Additionally, genomic RNA was encapsidated into virions in N. sylvestris protoplasts. Because the 5'NTR permitted efficient replication and production of replicase proteins, these findings suggest that the replicase of ORSV is responsible for the defect in cell-to-cell movement of ORSV in N. sylvestris .     

Tobamovirus replicase coding region is involved in cell-to-cell movement

Tobacco mosaic virus (TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126- and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.     

Phosphorylation and/or Presence of Serine 37 in the Movement Protein of Tomato Mosaic Tobamovirus Is Essential for Intracellular Localization and Stability In Vivo

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

The Tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108L-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108L-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108L-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.