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.     

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.     

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cucumber mosaic virus movement protein affects sugar metabolism and transport in tobacco and melon plants

In addition to its influence on plasmodesmal function, tobacco mosaic virus movement protein (TMV‐MP) causes an alteration in carbon metabolism in source leaves and in resource partitioning among the various plant organs. The present study was aimed at characterizing the influence of cucumber mosaic virus (CMV)‐MP on carbohydrate metabolism and transport in both tobacco and melon plants. Transgenic tobacco plants expressing the CMV‐MP had reduced levels of soluble sugars and starch in their source leaves and a significantly reduced root‐to‐shoot ratio in comparison with control plants. A novel virus‐vector system was employed to express the CMV‐coat protein (CP), the CMV‐MP or the TMV‐MP in melon plants. This set of experiments indicated that the viral MPs cause a significant elevation in the proportion of sucrose in the phloem sap collected from petioles of source leaves, whereas this sugar was at very low levels or even absent from the sap of control melon plants. The mode by which the CMV‐MP exerts its effect on phloem‐sap sugar composition is discussed in terms of possible alterations in the mechanism of phloem loading.     

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.     

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.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

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.     

Intracellular trafficking of Potato leafroll virus movement protein in transgenic Arabidopsis

Intracellular trafficking of viral movement proteins (MPs) in plants has mainly been studied using Tobacco mosaic virus MP30 (TMV MP30) as a model system. Because of the limitations of TMV MP30 expression in Arabidopsis thaliana, these studies have mostly been restricted to tobacco plants. Here we present data on the analysis of transgenic Arabidopsis plants expressing Potato leafroll virus 17-kDa movement protein (MP17) fused to green fluorescent protein. MP17 localizes to secondary branched plasmodesmata (PD) in source but not to simple PD in sink tissues, where MP17 is believed to be degraded by proteolysis. To unravel the intracellular transport path of MP17, we analyzed the relevance of the cytoskeleton and of the secretory pathway on MP17 targeting. To this end, a new incubation system for in vivo analysis of immediate and long-term responses of whole Arabidopsis plants to inhibitor treatments was established. Microscopic and histochemical analysis showed that MP17 is targeted to PD in an actin- and endoplasmic reticulum-Golgi-dependent manner. In contrast, degradation of MP17 in sink tissues required intact microtubules and occurred at 26S proteasomes. Interestingly, inhibition of the 26S proteasome led to aggregation of MP17 in aggresome-like structures. Formation of these structures could be inhibited by colchicine, as was shown for aggresomes in mammalian cells.     

Alteration in carbon partitioning induced by the movement protein of tobacco mosaic virus originates in the mesophyll and is independent of change in the plasmodesmal size exclusion limit

The influence of the 30 kDa movement protein of tobacco mosaic virus (TMV-MP) on carbon partitioning in trans-genie tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the TMV-MP was investigated. Using reciprocal grafting of transgenic tobacco plants expressing this movement protein and vector control plants, as well as transgenic tobacco plants expressing the TMV-MP in phloem cells only, we showed that the interactive site involved in carbon allocation to roots is localized to the mesophyll tissue. Biomass partitioning experiments conducted on transgenic plants, in which various deletion mutant forms of the TMV-MP (two of which included deletions in the domain responsible for increasing the size exclusion limit) were expressed, revealed that the TMV-MP exerts its influence on carbon allocation via a mechanism that is completely independent of the TMV-MP-induced increase in the plasmodesmal size exclusion limit. Furthermore, small N- and C-terminal deletions in the MP revealed the complexity of the interactions likely to be involved between the MP and an endogenous regulatory mechanism. We propose that the TMV-MP interferes with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.     

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.     

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.     

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.