Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Effects of calreticulin on viral cell-to-cell movement

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.     

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.     

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.     

Targeting and modification of prokaryotic cell–cell junctions by tobacco mosaic virus cell‐to‐cell movement protein

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.     

Mapping of Epitopes of the Recombinant Movement Protein of the Tobacco Mosaic Virus with the Use of Monoclonal Antibodies

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. MP fulfils its function while interacting with host cell factors on the whole way of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfilling the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MP_r) were obtained. MAs were tested by the immunoblotting and ELISA with the use of deletion variations of MP_r. The epitopes of TMV MP_r that recognize specific MAs were determined.     

Epitope mapping of the recombinant movement protein of the tobacco mosaic virus using monoclonal antibodies

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. The MP fulfills its function while interacting with host cell factors over the whole path of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfillment of the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by immunoblotting and ELISA with the use of deletion variants of MPr. The epitopes of TMV MPr that recognize specific MAs were determined.     

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.     

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.     

Interaction of tomato mosaic virus movement protein with tobacco RIO kinase

Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata. To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method. One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p. We isolated a full-length cDNA by RT-PCR. The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively. In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus. NtRIO had autophosphorylation activity and phosphorylated ToMV MP. Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO. The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.     

Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein.

A gene encoding a temperature-sensitive mutant (MPP154A) of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) was transformed into Nicotiana tabacum cv Xanthi. Transgenic plants expressing the MPP154A gene complemented local and systemic movement of an MP-defective mutant of TMV (U3/12MPfs) at the permissive temperature of 24 degrees C but not at 32 degrees C, the nonpermissive temperature. A microinjection procedure was used to investigate the effects of the modified TMV MP on plasmodesmatal size-exclusion limits. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran), with an average molecular mass of 9.4 kilodaltons, was detected between leaf mesophyll cells of the transgenic plants at 24 degrees C; however, no movement of either 3.9-kilodalton or 9.4-kilodalton F-dextrans was detected when the transgenic plants were held for 6 hours (or longer) at 32 degrees C. When these plants were shifted back to 24 degrees C for 6 hours, cell-to-cell movement of the F-dextrans was again observed. Accumulation of MPP154A was not affected by the temperature regime, nor was the subcellular distribution of the MP altered. These results are consistent with a change in the protein conformation of MPP154A at the nonpermissive temperature, which gives rise to a protein that fails to modify the molecular size-exclusion limits of plasmodesmata to the same extent as wild-type MP. Surprisingly, at 32 degrees C, movement of the F-dextrans was inhibited in transgenic plants expressing the wild-type MP gene; however, the inhibition was transient and was no longer detected after 48 hours at this elevated temperature. This transient inhibition of plasmodesmatal function was alleviated with Sirofluor, an inhibitor of callose ([1----3]-beta-D-glucan) synthesis. This result provides experimental evidence that callose deposition is involved in regulating the molecular size-exclusion limit of plasmodesmata in plants. Sirofluor had no effect on the inhibition of F-dextran movement at 32 degrees C in plants expressing the MPP154A gene, indicating that callose formation was not responsible for the failure of the temperature-sensitive mutant protein to alter the size-exclusion limit of plasmodesmata.     

Role of P30 in replication and spread of TMV

The P30 movement protein (MP) of tobacco mosaic virus is essential for distribution of sites of replication within infected cells and for cell–cell spread of infection. MP is an integral membrane protein and in early and mid-stages of infection causes severe disruption of the cortical endoplasmic reticulum (ER). MP also associates with microtubules, and in late stages is targeted for degradation by the 26S proteosome. During these stages, the ER regains its normal pre-infection configuration. Viral RNA is associated with ER and microtubules in the presence of MP. The MP is phosphorylated and mutation of the phosphorylated amino acid reduced association of MP with the ER, plasmodesmata, and microtubules, and altered the stability of the MP. The nature of the association of MP with vRNA and ER and microtubules, and the role of phosphorylation of MP in each of these functions, if any, remains to be determined.     

Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.     

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.     

Tobacco mosaic virus movement protein enhances the spread of RNA silencing

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.     

Dye-coupling in Tobacco Mesophyll Cells Surrounding Growing Tobacco Mosaic Tobamovirus-induced Local Lesions

Some factors related to cessation of the movement of tobacco mosaic tobamovirus (TMV) in the late phase of a defence response were examined. Mesophyll cells surrounding the local lesions induced by TMV in N. tabacum cv. Xanthi‐nc were micro‐injected with fluorescent dye 2, 3 and 7 days post‐inoculation. At 7 days post‐inoculation, twelve out of 20 injections into cells adjacent to the lesion, showed the expected dye‐coupling (outflow of fluorescent dye from injected cell to adjacent ones via plasmodesmata) whereas 17–20 out of 20 injections were successful in other cases. Callose inhibitor (tunicamycin), dark treatment and incubation of plants with ascorbic acid, which play a role in blocking plasmodesmata or induction of defence responses, did not seem to have an effect on lesion growth. These data imply that defects in the plasmodesmal function, although not total, may account for the formation of late local defence reaction together with other factors that restrict viral spread in the continuous cell‐to‐cell mode in mesophyll tissue.     

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.