Tobacco Mosaic Virus Movement Protein Research Articles

Distribution of TMV movement protein in single living protoplasts immobilized in agarose.

Recent studies of the tobacco mosaic virus (TMV) P30 movement protein (MP) fused with green fluorescent protein (GFP) during TMV infection described the involvement of elements of the cytoskeleton and components of the endoplasmic reticulum (ER) in the intracellular trafficking of MP:GFP from the sites of synthesis in the cytoplasm to plasmodesmata. To examine in real-time the pattern of synthesis, accumulation and degradation of MP:GFP, we developed a method to immobilize protoplasts in agarose such that they are maintained alive for extended periods of time. The pattern of MP:GFP accumulation in single living protoplasts visualized by confocal laser scanning microscopy (CLSM) was parallel to that previously described in a population of protoplasts harvested at different times post-infection. Additionally, a network of weakly fluorescent filaments, which are apparently different from microtubules, was observed to surround the nucleus and these filaments were associated with fluorescent bodies (previously identified as ER-derived structures). Later in infection, the fluorescent bodies increased in size and coalesced to form larger structures that accumulated near the periphery of the cells while highly fluorescent non-cortical filaments were observed distributed in the cytoplasm. The putative involvement of these filaments in targeting the fluorescent bodies to the periphery of the cell is discussed. Studies of single, embedded protoplasts make it possible to observe changes in amount and subcellular localization of viral and other proteins.

Domains of the TMV movement protein involved in subcellular localization.

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9-11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49-51 may be involved in co-alignment of the protein with microtubules. The region around residues 88-101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.

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.

Changing Patterns of Localization of the Tobacco Mosaic Virus Movement Protein and Replicase to the Endoplasmic Reticulum and Microtubules during Infection

Changing Patterns of Localization of the Tobacco Mosaic Virus Movement Protein and Replicase to the Endoplasmic Reticulum and Microtubules during Infection

Detection of Tobacco Mosaic Virus Movement Protein in Association with Tobacco Nuclei Isolated from Intact and Detached Leaves

AbstractThe movement protein (MP) of tobacco mosaic virus was observed to cofractionate with nuclei from intact and detached tobacco (Nicotiana tahacum cv. Xanthi‐nn) leaves. When purified nuclei were treated with proteinase K, MP disappeared indicating that MP is localized close to but not inside nuclei. Moreover, the amount of MP was showti to increase greatly in nuclei isolated from detached leaves, thus facilitating the detection of MP. Accumulation close to nuclei was strongest early in infection, results indicating that it may be an early step in the pathway of MP from cell cytoplasm to plasmodesmata. Other TMV‐specific proteins were not detected in the nuclei fraction.

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.

Phloem-Specific Expression of the Tobacco Mosaic Virus Movement Protein Alters Carbon Metabolism and Partitioning in Transgenic Potato Plants.

The tobacco mosaic virus movement protein (TMV-MP) has pleiotropic effects when expressed in transgenic tobacco (Nicotiana tabacum) plants. In addition to its ability to increase the plasmodesmal size-exclusion limit, the TMV-MP alters carbohydrate metabolism in source leaves and dry matter partitioning between the various plant organs. In the present study the TMV-MP was expressed under the control of a phloem-specific promoter (rolC), and this system was employed to further explore the potential sites at which the TMV-MP exerts its influence over carbon metabolism and transport in transgenic potato (Solanum tuberosum) plants. Immunohistochemical analyses indicated that the TMV-MP was localized mainly to phloem parenchyma and companion cells. Starch and sucrose accumulated in source leaves of these plants to significantly higher levels compared with control potato lines. In addition, the rate of sucrose efflux from excised petioles was lower compared with control plants. Furthermore, under short-day conditions, carbon partitioning was lower to the roots and higher to tubers in rolC plants compared with controls. These results are discussed in terms of the mode(s) by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation.

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In this study, microinjection protocols were used to directly examine cell-to-cell trafficking of CMV viral RNA in these resistant plants. CMV RNA fluorescently labeled with the nucleotide-specific TOTO-1 iodide dye, when coinjected with unlabeled CMV 3a movement protein (MP), moved rapidly into the surrounding mesophyll cells in mature tobacco leaves of vector control and untransformed plants. Such trafficking required the presence of functional CMV 3a MP. In contrast, coinjection of CMV 3a MP and CMV TOTO-RNA failed to move in transgenic resistant plants expressing the CMV truncated replicase gene. Furthermore, coinjection of 9.4-kDa fluorescein-conjugated dextran (F-dextran) along with unlabeled CMV 3a MP resulted in cell-to-cell movement of the F-dextran in control plants, but not in the transgenic plants. Similar results were obtained with viral RNA when the 30-kDa MP of tobacco mosaic virus (TMV) was coinjected with TMV TOTO-RNA into replicase-resistant transgenic tobacco expressing the 54-kDa gene sequence of TMV. However, in these transgenic plants, the TMV-MP was still capable of mediating cell-to-cell movement of itself and the 9.4-kDa F-dextran. These results indicate that an inhibition of cell-to-cell viral RNA trafficking is correlated with replicase-mediated resistance. This raises the possibility that the RNA-2 product is potentially involved in the regulation of cell-to-cell movement of viral infectious material during CMV replication.

Tissue-Specific Expression of the Tobacco Mosaic Virus Movement Protein in Transgenic Potato Plants Alters Plasmodesmal Function and Carbohydrate Partitioning.

Transgenic potato (Solanum tuberosum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) under the control of the promoters from the class I patatin gene (B33) or the nuclear photosynthesis gene (ST-LS1) were employed to further explore the mode by which this viral protein interacts with cellular metabolism to change carbohydrate allocation. Dye-coupling experiments established that expression of the TMV-MP alters plasmodesmal function in both potato leaves and tubers when expressed in the respective tissues. However, whereas the size-exclusion limit of mesophyll plasmodesmata was increased to a value greater than 9.4 kD, this size limit was smaller for plasmodesmata interconnecting tuber parenchyma cells. Starch and sugars accumulated in potato leaves to significantly lower levels in plants expressing the TMV-MP under the ST-LS1 promoter, and rate of sucrose efflux from petioles of the latter was higher compared to controls. It is interesting that this effect was expressed only in mature plants after tuber initiation. No effect on carbohydrate levels was found in plants expressing this protein under the B33 promoter. These results are discussed in terms of the mode by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation, and the possible role of plasmodesmal function in controlling these processes.

Movement of a Barley Stripe Mosaic Virus Chimera with a Tobacco Mosaic Virus Movement Protein

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNAβ replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs α and γ, together with the hybrid RNAβ transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves ofNicotiana benthamianaandChenopodium amaranticolor,which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions onC. amaranticolorwere influenced by the foreign MP gene. However, the hybrid virus failed to infect barley,N. tabacum(var. Samsun), andN. clevelandii,the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.

Tobacco Mosaic Virus Movement Protein Associates with the Cytoskeleton in Tobacco Cells

Tobacco Mosaic Virus Movement Protein Associates with the Cytoskeleton in Tobacco Cells

Tobacco Mosaic Virus Movement Protein: Mediated Protein Transport between Trichome Cells

Tobacco Mosaic Virus Movement Protein: Mediated Protein Transport between Trichome Cells

Tobacco mosaic virus movement protein-mediated protein transport between trichome cells.

Tobacco mosaic virus movement protein (TMV MP) is required to mediate viral spread between plant cells via plasmodesmata. Plasmodesmata are cytoplasmic bridges that connect individual plant cells and ordinarily limit molecular diffusion to small molecules and metabolites with a molecular mass up to 1 kD. Here, we characterize functional properties of Nicotiana clevelandii trichome plasmodesmata and analyze their interaction with TMV MP. Trichomes constitute a linear cellular system and provide a predictable pathway of movement. Their plasmodesmata are functionally distinct from plasmodesmata in other plant cel types; they allow cell-to-cell diffusion of dextrans with a molecular mass up to 7 kD, and TMV MP does not increase this size exclusion limit for dextrans. In contrast, the 30-kD TMV MP itself moves between trichome cells and specifically mediates the translocation of a 90-kD beta-glucuronidase (GUS) reporter protein as a GUS::TMV MP fusion. Neither GUS by itself nor GUS in the presence of TMV MP moves between cells. These data imply that a plasmodesmal transport signal resides within TMV MP and is essential for movement. This signal confers selectivity to the translocated protein and cannot function in trans to support movement of other molecules.

Multiple serine phosphorylation sites on the 30 kDa TMV cell‐to‐cell movement protein synthesized in tobacco protoplasts

p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro. Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61-114 and residues 212-231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.

Evidence for proteolytic processing of tobacco mosaic virus movement protein in Arabidopsis thaliana.

Two ecotypes of Arabidopsis thaliana were transformed with the gene encoding tobacco mosaic virus (TMV) movement protein (P30). P30 accumulated largely in a subcellular fraction containing cell wall components and as a soluble protein. The protein migrated in denaturing gels with an M(r) of 30K, significantly faster than P30 (M(r) approximately 34K) accumulating after expression in transgenic tobacco, Escherichia coli or Spodoptera frugiperda cells, or after virus multiplication in tobacco. The P30 from A. thaliana infected with TMV for 14 days comigrated with that from E. coli, but that from A. thaliana infected for 49 days was of the smaller size. The use of antisera specific for the N- or C-termini of P30 showed that in A. thaliana P30 was proteolytically processed at the N-terminus, a region essential for P30 function. The failure of these plants to complement a TMV P30 mutant indicated that processed P30 was nonfunctional, although the processing was not so rapid that it prevented the development of systemic infections with wild type TMV. The absence of detectable P30 phosphorylation in A. thaliana demonstrated that phosphorylation was not essential for movement protein function and suggested that this species may use proteolytic cleavage of the N-terminus as an alternative strategy to tobacco for deactivating P30.

Involvement of Residues Within Putative alpha Helix Motifs in the Behavior of the Alfalfa and Tobacco Mosaic Virus Movement Proteins

The movement proteins (MPs) of both alfalfa mosaic virus (AMV) and tobacco mosaic virus (TMV) have a 15-amino acid putative α helix motif that is part of a larger domain involved in the cell wall (CW) localization of these proteins. These α helices have common features since they are amphipathic and contain two clusters of acidic amino acids, EE and DE. Mutations were introduced into these helices to investigate their role in CW localization and in the activity of the MPs. Results showed that both these motifs are involved in the CW localization of the MPs, although through different mechanisms : whereas the helical structure and the EE cluster of the AMV-MP were required for optimum CW localization, the DE cluster of the TMV-MP, but not the helical structure, was involved in this process. Chimeric proteins resulting from an exchange of the α helices between MPs showed that these sequences did not function in the complementary background. Moreover, the region around aa 61 of the TMV-MP is necessary for protein stability. Transgenic tobacco plants expressing a mutated AMV-MP in which the α helix was deleted or had its structure destroyed exhibited an abnormal development and a modified morphology. In parallel, a similar mutation of the TMV-MP yielded a nonfunctional protein that still accumulated in the CW but that did not compete with the viral MP during infection.

A defective movement protein of TMV in transgenic plants confers resistance to multipleviruses whereas the functional analog increases susceptibility

Transgenic tobacco plants that express a gene encoding a defective mutant of the tobacco mosaic virus (TMV) movementprotein which are known to be resistant to several tobamoviruses were inoculated with viruses from different taxonomic groups to determine the breadth of resistance. There were significant delays in the time of appearance of disease symptoms and/or there was reduced systemic accumulation of virus in upper leaves of plants inoculated with tobacco rattle tobravirus, tobacco ringspot nepovirus, alfalfa mosaic alfamovirus, peanut chlorotic streak caulimovirus, and cucumber mosaic cucumovirus. Conversely, tobacco plants that express a gene encoding the functional tobacco mosaic virus wild-type movement protein accelerated symptom development, enhanced the severity of symptom formation, and/or increased the accumulation of these viruses and, additionally, TMV. Our results indicate that there are similar functions among the movement proteins of a number of plant viruses despite the apparent lack of sequence similarity between them.

Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant.

Tobacco mosaic virus (TMV) which contains the movement protein (MP) of odontoglossum ringspot tobamovirus (ORSV) in place of the TMV MP systemically infects orchids but causes local infection in tobacco unless the carboxy-terminal 48 amino acids of the MP are deleted (C. A. Holt, C. A. Fenczik, S. J. Casper, and R. N. Beachy; Virology, in press, 1995). Frameshift mutations were created within the 3' ends of the MP gene that led to truncations of the ORSV MP by 11, 19, 28, 37, and 48 amino acids; each of the mutant MP genes was inserted into the cloned cDNA of TMV in place of the TMV MP and infectious transcripts were produced. Virus containing mutant MPs were used to infect vanilla orchids, a systemic host of ORSV, and tobacco plants. Removal of 11 amino acids from the ORSV MP prevented spread of the chimeric virus in orchids while restoring the ability to cause a systemic infection on tobacco. Further deletions of the MP affected the size of virus-induced necrotic local lesions on tobacco cv. Xanthi NN and the systemic spread and accumulation of virus in cv. Xanthi nn, a systemic host of TMV. However, each virus replicated to equivalent levels in protoplasts. A mechanism by which the ORSV MP limits the spread of the chimeric virus is proposed.

A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants

A chimeric gene encoding a dysfunctional tobacco mosaic virus (TMV) movement protein (MP) mutant lacking amino acids 3, 4 and 5 (MPΔ3–5), was expressed in transgenic Nicotiana tabacum Xanthi and Xanthi NN plants. Immunogold labeling studies of tissues from transgenic plants indicated that while wild‐type MP accumulated in the plasmodesmata, MPΔ3–5 did not. Tissue fractionation studies confirmed that only a low level of the mutant MP accumulated in the cell wall‐enriched fraction compared with the accumulation of the wild‐type MP. Dye coupling studies showed that MPΔ3–5 enabled the movement between leaf mesophyll cells of a fluorescently labeled dextran of 3 kDa, while 9.4 kDa molecules failed to move. In contrast, in transgenic plants expressing the wild‐type MP gene the 9.4 kDa probe did move from cell to cell. Seedlings from self‐fertilized transgenic plants were inoculated with TMV and observed for disease symptoms. Transgenic Xanthi NN plants that expressed the MPΔ3–5 gene developed fewer and smaller necrotic local lesions compared with control plants following inoculation with TMV. Transgenic Xanthi nn plants were delayed in the development of systemic symptoms. Inoculating the transgenic plants with TMV‐RNA, and the tobamo‐viruses TMGMV and SHMV, essentially produced the same results, i.e. inhibition of disease development. These results demonstrate that transgenic plants expressing an inactive MP can inhibit virus disease spread presumably by interfering with its cell‐to‐cell movement.