What is the significance of the tobacco mosaic virus

N, nucleus. Tobacco mosaic virus and the very closely related Tomato mosaic virus ToMV use their parental genomes to synthesize complementary negative strands which serve as templates for the synthesis of progeny full-length positive strands and subgenomic mRNAs containing MP and CP open reading frames ORFs; Ishikawa and Okada, ; Ishibashi et al.

The and kDa proteins contain methyltransferase and helicase domains, while the kDa protein alone contains the C-terminal domain encoding an RNA-dependent RNA polymerase. The and kDa proteins together will be referred to as the replication proteins in this review. Reichel and Beachy , using transgenic plants expressing an ER-targeted GFP, determined that the ER formed large cortical aggregates at reticulate vertices and fewer membrane tubules during TMV accumulation, but returned to a normal structure after replication ended.

However, later studies with ToMV using both fluorescence microscopy and biochemical fractionation methods determined that the replication proteins and replicase activity were associated predominantly with the vacuolar membrane, although they also showed some localization and activity with other less defined membrane fractions which included the ER Hagiwara et al.

Interestingly, ToMV can replicate in cells that are vacuole-diminished Nishikiori et al. Clearly, additional work is necessary to determine which membranes are essential for tobamovirus accumulation Figure 1. Regardless of the membrane used for tobamovirus accumulation, it is clear that a characteristic VRC is not uniformly induced by tobamoviruses. Early fluorescence localization studies determined that for strains of TMV the size of the VRCs varied and was positively correlated with the level of disease observed Liu et al.

Recently, however, it was determined that silencing expression of the gamma subunit of ATP synthase, a nuclear-encoded chloroplast protein, resulted in smaller but more numerous VRCs and severe disease symptoms Bhat et al. Thus, the size of the VRC is not a perfect indicator of disease intensity and the number of VRCs may influence this phenotype. The form of inclusions induced by tobamoviruses is correlated with differences in the replication protein sequences Liu et al.

Domain s within the kDa protein necessary for inclusion body formation are not identified, however, it is known that the helicase domain when expressed alone is able to form octomers in vitro Goregaoker and Culver, and thus may be a domain important for this activity. The amino acid protein includes the methyltransferase and non-conserved bridge domain that previously was determined to influence the RNA silencing suppression function of this protein.

There is unpublished data indicating that the methyltransferase domain alone can form inclusions Knapp et al. More work is needed to further identify the domains responsible for inclusion formation and the relevance of inclusion formation to tobamovirus physiology. Ectopically expressed TMV MP fused with fluorescent reporter proteins also can form cytoplasmic inclusion bodies Heinlein et al.

These small inclusions are similar to those visualized in the cortical periphery during infection with tobamoviruses expressing an MP-GFP fusion Padgett et al. In this regard, it will be important to determine if multiple types of inclusions are formed independently during infection or are always part of a continuum, with progeny inclusions appearing from parental inclusions.

The host proteins within VRCs or inclusions that contain them are not fully identified. Ding and R. Nelson, personal communication. The function of this protein in X-bodies is unknown, but perhaps it could be to aid the degradation of body components analogous to the suggested function of microtubules during TMV MP turnover Kragler et al.

The host translation factor, elongation factor 1A EF-1A , is present in the membrane-associated fraction where viral replicase activity was observed Osman and Buck, ; Watanabe et al. EF-1A has additional activities beyond supporting translation including forming complexes with tubulin and actin, the actin interaction possibly linking the cytoskeleton to protein synthesis, and ubiquitin-mediated degradation Durso and Cyr, ; Gonen et al.

However, down-regulation of EF-1A through virus-induced gene silencing resulted in the reduced size of green fluorescent lesions induced by TMV-expressing GFP, but no reduction in lesion numbers or translation activity in the silenced leaves Yamaji et al. This result suggests that the function of EF-1A is not for translation or virus accumulation, but for virus movement that may, in some manner, be linked to the cytoskeleton Figure 1.

Tobamovirus multiplication 1 TOM1 is a predicted multi-pass transmembrane protein required for tobamovirus accumulation Ishikawa et al. This observation is associated with the finding that accumulation of the ToMV replication proteins in membrane-free soluble fractions was lower for plants over-expressing TOM1 compared with those not over-expressing this protein Hagiwara-Komoda et al.

This result indicates that the level of the soluble form or the ratio of soluble and membrane-bound forms of the replication proteins is critical for normal virus accumulation. It was hypothesized that the soluble form is important for RNA silencing suppressor activity and it was shown that the loss of suppressor activity is correlated with diminished accumulation of the virus Kubota et al. TOM1 interacts with the helicase domain of the kDa protein from the related tobamovirus, Tobacco mosaic virus-Cg crucifer-infecting virus , in a yeast two-hybrid screen Yamanaka et al.

This interaction was shown to be with the helicase core region based on predictions from the crystal structure of the helicase domain Nishikiori et al. The replication proteins from ToMV and TOM1 share similar subcellular fractionation pattern in extracts from infected BY-2 cells, residing mostly in the tonoplast-containing fractions, but also in fractions with other membranes, including the ER Hagiwara et al.

It is hypothesized that TOM1 forms a link between the host membrane in which it resides and the tobamovirus replication proteins Figure 1. This interaction is likely important for VRC formation, but the co-localization of TOM1 and tobamovirus replication proteins in live cells has not been reported.

For TMV to establish a systemic infection, the virus or its components must move within a cell to establish an infection site, multiply and finally position for movement to the next cell. These complexes may change their form and constituents with time. During initial infection, granules containing vRNA anchor to cortical ER and move to cortical ER vertices and the perinuclear ER where virus replication and translation occurs Reichel and Beachy, ; Christensen et al.

Indeed, vRNA has been visualized in the perinuclear bodies by bimolecular fluorescence complementation using a modified sequence-specific RNA-binding protein, Pumilio1, or by classical in situ RNA labeling Tilsner et al.

However, considering that most of the ribosome-containing, or rough, ER is present in the perinuclear region Carrasco and Meyer, it is likely that this location, or a cortical ER vertex also containing ribosomes, is best suited for virus protein synthesis. Neither cytochalasin D nor latrunculin B LatB treatment, both microfilament antagonists, affect granule formation suggesting that microfilaments are not involved in this initial activity Christensen et al.

However, disruption of microfilaments results in granules hovering in the cortical ER, suggesting microfilaments help transport the granules in the cell. In contrast, depolymerizing microtubules does not stop vRNA granule movement along the tubular cortical ER Christensen et al. TMV replication occurs in association with ER and other membranes and both the MP and the replication proteins associate intrinsically or through a protein linker with membranes Brill et al.

The actual pathway used by the virus, however, likely includes ER since that membrane is present in early- and late-formed virus inclusions by fluorescence microscopy and EM , with early forming inclusions paired at the cell wall e. Much information is available on the movement of inclusion bodies containing the viral replication proteins. Several laboratories pursued EM-based immunocytolocalization studies of TMV infections with antibody against the replication proteins Hills et al. They noted that the structure of the inclusions likely changed during development, going from smoothly granular to containing electron-dense rope-like structures, composed at least partly of kDa protein, in a ribosome-rich matrix.

Saito et al. Near the infection front the inclusion bodies were paired on either side of the cell wall and contained both the replication proteins and MP, while four to six cells back from the front the bodies were not paired, had moved away from the cell wall and only contained the replication proteins.

Through fluorescence microscopy of cells near the infection front, Tilsner et al. Motionless small fluorescent bodies in the cell were detected at 12 h post-inoculation and these bodies were moving by 14 h post-inoculation when tracking TMV expressing an MP-GFP fusion: a period when both MP and the replication proteins would co-localize Kawakami et al.

Movement of the fluorescent inclusions early in infection, when both replication proteins and MP would be present, was aligned with microfilaments, and through pharmacological and gene-silencing studies, the inclusions also referred to as VRCs were shown to require these intact microfilaments for their intracellular movement Kawakami et al. The degradation of the microfilaments was not sufficient to decrease virus accumulation to levels that would prevent virus movement or VRC formation and thus the influence of microfilaments on TMV intracellular movement was not confounded by a significant inhibition of virus replication Liu et al.

Treatment with a general myosin motor inhibitor, 2,3-butanedione monoxime BDM , impaired the intracellular movement of VRCs Kawakami et al. Boyko et al. Regarding microfilaments, early studies indicated an association of TMV MP with rhodamine-conjugated phalloidin microfilaments after probing cells with polyclonal antibody against the MP and fluorescein-conjugated secondary antibody McLean et al.

Wright et al. However, a later study determined that the MP-GFP expressed during virus infection was not observed to co-align with fluorescently labeled microfilaments Hofmann et al. Thus, during virus infection the trafficking of the MP within the cell likely requires intact ER and may require microfilaments and microtubules Figure 1 , although evidence against a direct action for microfilaments exists and microtubules may not be in an intact form or the required microtubule array is unusual in that it is impervious to certain pharmacological agents Seemanpillai et al.

Their independent transport may have physiological relevance. Liu, and R. Whether the kDa protein directly interacts with microfilaments or myosin XI-2 requires further study. If there is no direct interaction between these proteins, trafficking of virus proteins may be through interaction of myosin XI-2 with host components associated with a virus-host protein complex or through the creation of a bulk flow network of cytoplasmic constituents i.

Considering that the viral replication proteins may transport independently of the MP or other viral components to complete their functions, additional studies of their ectopic transport in relationship to their transport during virus infection are needed. The difficulty in pursuing studies on the viral replication proteins is that no fusion between the replication proteins and a fluorescent marker has been developed that yields a viable virus.

This needs to be addressed for further progress to occur. Transient expression of the MP fused with fluorescent markers results in the formation of inclusions that associate with RNA and associate with and traffic along the ER, perhaps interacting with a microtubule scaffold necessary for movement Sambade and Heinlein, Both microfilament and microtubule antagonists inhibited intracellular transport of the MP-fluorescent marker fusion, the results from the latter treatment using aminoprophos-methyl being a different finding from many during virus infection where MP-GFP fusion movement was not impeded by microtubule antagonists.

To complicate this situation further, additional research determined that neither oryzalin or aminoprophos-methyl, both antagonists of microtubules, nor LatB, a microfilament antagonist, inhibited the formation of MP-GFP inclusions or their localization to the cell periphery or PD in cultured cells or leaves Prokhnevsky et al. These apparently conflicting results highlight the difficulty interpreting findings from pharmacological studies.

Clearly, additional work is required to determine, in real time and through methods using non-pharmacological techniques, the influence of the cytoskeleton on transiently expressed MP intracellular trafficking.

Assuming MP trafficking independent of the VRC has physiological relevance, findings from these additional studies would provide further insight into the mechanism of TMV intracellular movement. Some work investigating the influence of MP transport in the absence of pharmacological treatment has been published.

For example, Kotlizky et al. This suggests that the microtubule binding domain resides in a different location from the region important for PD localization and supports the pharmacological studies indicating that MP PD localization and initial virus spread requires more than microtubule association by the MP.

As for intracellular movement, our understanding of TMV intercellular movement is fragmented. PD are bounded by the plasma membrane and contain a cytoplasmic sleeve between this membrane and intact ER, the ER referred to as the desmotubule in this tissue Lucas et al. Callose is present in the neck region of the PD Northcote et al.

Actin and myosin are among multiple protein components in the cytoplasmic sleeve Fernandez-Calvino et al. This presents an impediment to virus movement because virus structures that are hypothesized to move between cells require a far larger SEL. The MP itself also can move between cells when ectopically expressed reviewed in Niehl and Heinlein, These findings, in total, suggest a mechanism for virus spread where TMV MP opens PD through its microfilament severing activity, mimicking the phenotype induced with microfilament antagonists Figure 1.

Su et al. This mutant MP, which also has severing activity, fragments microfilaments in the cytoplasm Su et al. The kDa protein fused with GFP has not been reported to move between cells during ectopic expression, suggesting that VRC intercellular movement requires expression of additional viral proteins likely the MP or the presence of the vRNA.

ANK has multiple activities, including binding to and delivering chloroplasts to their destination, supporting disease resistance against bacteria and virus challenge and participating in reactive oxygen scavenging, but it does not have callose degrading activity.

However, in many instances cell biological studies to observe the interaction between the MP and host proteins within a live cell have not been conducted to further determine the location where the interaction may influence intercellular spread. Brandner et al. EB1a-GFP sub-cellular localization during TMV infection was altered from end labeling comet-like structures representing growing microtubules to labeling the length of microtubules.

This unexpected re-localization of the EB1a protein at the infection front was correlated with an inhibition of virus intercellular spread. Ouko et al. These findings support those using a mutant strain of TMV whose temperature sensitive intercellular movement is correlated with the temperature sensitive localization of MP with microtubules Boyko et al. Additionally, a virus expressing a modified MP with limited affinity for microtubules displayed enhanced intercellular spread Gillespie et al.

These and other findings from studies of a microtubule-binding protein, MPB2C, which binds to the MP and when overexpressed has a negative effect on intercellular movement of a related tobamovirus Ruggenthaler et al.

These authors also determined that overexpression of CDC48 in infected tissue inhibited virus spread. Thus, it appears that removal of the MP from the ER at the infection front and its movement to the microtubules through CDC48 activity is directed toward processing and possibly, degrading, the MP Figure 1.

Microfilaments have been shown to be important for TMV intercellular movement, but the interpretation of their involvement in this activity is evolving.

Findings from early studies using a GFP-labeled virus showed that disruption of microfilaments, through pharmacological methods or by silencing actin, inhibited sustained 2 days and beyond post-inoculation virus intercellular movement Kawakami et al.

This inhibition in sustained intercellular spread was not associated with a decrease in virus accumulation per cell that would affect virus movement or prevent VRC formation Liu et al. In addition, the sustained virus intercellular movement was not correlated with VRC size Liu et al.

This movement required myosin motor activity and specifically myosin XI-2 motor activity Kawakami et al. Surprisingly, intercellular movement of the related tobamovirus, TVCV, was unaffected by disruption of microfilaments or silencing of any myosin studied to date Harries et al. During this time, it also was shown that TMV was not inhibited in spread early after LatB treatment i. They concluded that this disruption in virus movement was primarily due to a loss of membrane fluidity caused by the ABD2-GFP marker and that TMV intercellular movement was predominantly influenced by membrane diffusion characteristics.

Thus, results from studies of both TVCV and early TMV intercellular movement suggest membranes as the predominant vehicle controlling tobamovirus intercellular movement. These findings also support those from a previous study showing that both the viral replication proteins and MP are necessary to allow maximum diffusion through PD of GFP-fused probes representing soluble ER membrane-bound proteins Guenoune-Gelbart et al.

It was concluded that the results best support a model in which the virus complex, perhaps consisting of viral RNA, MP and other proteins, diffuses on the ER membrane within the PD from infected to uninfected cells driven by a concentration gradient Guenoune-Gelbart et al.

Harries et al. These cells would then modify their metabolism in preparation for the arrival of the virus. This stress would be signaled to the cells in advance of the virus spread and modify these cells to inhibit subsequent virus spread. This interpretation would also accommodate the finding that early TMV movement is unaffected by actomyosin inhibitors since it would take some time to signal in advance of the infection front to stop virus movement. In addition, it would explain the lack of effect of LatB on TVCV movement since the kDa protein, the homolog of the kDa protein of TMV, does not form intracellular inclusions that associate with microfilaments Harries et al.

A dominant negative synaptotagmin mutant caused depletion of endosomes and inhibited intercellular trafficking of the MP-GFP fusion Lewis and Lazarowitz, This hypothesis, suggesting a requirement for actomyosin-mediated vesicle trafficking of the VRC or VRC components from the wall membranes for sustained virus movement, can be evaluated through cell biological studies.

It is possible that an interaction between two host proteins and the TMV MP aid in transport of virus between cells Figure 1. Cell biological studies over the last 20 years have tremendously aided our understanding of TMV accumulation and spread. Without advanced molecular and biochemical technologies allowing virus and virus component labeling and advanced imaging hardware our understanding of the individual processes during virus spread would be diminished.

This said, some cell biological studies have yielded conflicting or controversial results. This is especially true of pharmacological studies and researchers must carefully control as many variables in these studies as possible. In addition, conclusions from pharmacological studies should be verified using other methods. Use of novel virus labeling techniques and advanced microscopes will allow further advances in this area.

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Tobacco mosaic virus TMV is named for one of the first plants in which it was found in the s. However, it can infect well over different species of plants. Symptoms associated with TMV infections: stunting mosaic pattern of light and dark green or yellow and green on the leaves malformation of leaves or growing points yellow streaking of leaves especially monocots yellow spotting on leaves distinct yellowing only of veins Some of the above symptoms can also be caused by high temperature, insect feeding, growth regulators, herbicides, mineral deficiencies, and mineral excesses.

Managing TMV No chemicals cure a virus-infected plant. Purchase virus-free plants. Remove all weeds since these may harbor TMV. Remove all crop debris from benches and the greenhouse structure. Set aside plants with the above symptoms and obtain a diagnosis. Discard infected plants. Disinfest tools by placing them in disinfectant for at least 10 min. Rinse thoroughly with tap water. Disinfest door handles and other greenhouse structures that may have become contaminated by wiping thoroughly with one of these materials.

Propagate plants via seed rather than vegetatively. Thoroughly wash hands after handling tobacco products or TMV-infected plants. Do not keep tobacco products in the pockets of clothing worn into the greenhouse.

Care should be taken to dispose of dead leaves and old plants, because dry, TMV-infected leaves can be blown around the greenhouse as 'dust' which can subsequently infect healthy plants if they are wounded.

Cross protection. Inoculation of a mild strain of the virus onto young plants can protect them from subsequent infection by more severe strains of TMV.

This is a well documented control strategy, called "cross protection," that is successfully applied in greenhouse operations.

Transgenic plants also offer alternative strategies for virus control see Biotechnology Figure Several tobacco and tomato cultivars have been bred to be genetically resistant to TMV. Genetic engineering techniques have provided scientists with the ability to express the TMV coat protein gene in transgenic tobacco and tomato plants. This control strategy can safeguard the plants from infection by closely related strains of the virus Figure Elimination of inoculum.

Under experimental conditions, it has been shown that TMV can be inactivated when workers dip their contaminated hands in milk prior to planting. This inexpensive technique greatly reduces the incidence of disease Figure Seedlings that are known to be susceptible should not be transplanted into soil that contains TMV-contaminated root or plant debris. Scouting for disease. During the growing season, infected plants should be dug up, bagged, and removed from the field. Rotation practices that include resistant plants or non host crops also should be employed to reduce the amount of inoculum in the field.

TMV can easily overwinter on the seed coat, thus providing an inoculum source for the next planting cycle. Both treatments will inactivate the virus that is on the seed coat, but should have little negative effect on seed germination. In , Martinus W. Beijerinck, of the Netherlands, put forth his concepts that TMV was small and infectious.

Furthermore, he showed that TMV could not be cultured, except in living, growing plants. This report, suggesting that 'microbes' need not be cellular, was to forever change the definition of pathogens.

In , Wendall Stanley was awarded the Nobel Prize for his isolation of TMV crystals, which he incorrectly suggested were composed entirely of protein.

Research by F. Bawden and N. Pirie, in England, during the same period correctly demonstrated that TMV was actually a ribonucleoprotein, composed of RNA and a coat protein. This discovery ushered in the modern era of molecular virology. TMV is known for several 'firsts' in virology, including the first virus to be shown to consist of RNA and protein, the first virus characterized by X-ray crystallography to show a helical structure Figure 7 , and the first virus used for electron microscopy Figure 6 , solution electrophoresis and analytical ultracentrifugation.

TMV also was the first RNA virus genome to be completely sequenced, the source of the first virus gene used to demonstrate the concept of coat protein mediated protection Figure 11 , and the first virus for which a plant virus resistance gene the N gene was characterized.

Today, TMV is still at the forefront of research leading to new concepts in transgenic technology for virus resistance and developing the virus to act as a 'work horse' to express foreign genes in plants for production of pharmaceuticals and vaccines.

Abel, P. Nelson, B. De, N. Hoffmann, S. Rogers, R. Fraley, and R. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science Ding, B. Intercellular protein trafficking through plasmodesmata.

Plant Mol.