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PubMed1997

Influence of Environmental Stress on Biomass Partitioning in Transgenic Tobacco Plants Expressing the Movement Protein of Tobacco Mosaic Virus.

The influence of various environmental factors on biomass partitioning between shoots and roots in transgenic tobacco (Nicotiana tabacum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) was investigated. TMV-MP-expressing transgenic plants exhibited a root-to-shoot ratio that was approximately 40% below that of transgenic vector control plants. When transgenic plants expressing the TMV-MP were subjected to water-stress conditions, the root-to-shoot ratio was increased to a value comparable to that of control plants subjected to the same water-stress treatment. Although the root-to-shoot ratio was increased by N or P deficiencies, the TMV-MP-induced alteration in biomass partitioning was not overcome. Surprisingly, under K+-deficient growth conditions, both TMV-MP-expressing and control plants exhibited reduced root-to-shoot ratios when compared with plants grown in the presence of sufficient K+. Furthermore, plant growth under K+-deficient conditions did not alleviate the influence of the TMV-MP over resource allocation to the roots. These results are discussed in terms of possible mechanisms by which stress signals could cause an alteration in biomass partitioning between shoots and roots in control and transgenic tobacco plants expressing the TMV-MP.

Journal Article↗
PubMed2001

An early tobacco mosaic virus-induced oxidative burst in tobacco indicates extracellular perception of the virus coat protein.

Induction of reactive oxygen species (ROS) was observed within seconds of the addition of exogenous tobacco mosaic virus (TMV) to the outside of tobacco (Nicotiana tabacum cv Samsun NN, EN, or nn) epidermal cells. Cell death was correlated with ROS production. Infectivity of the TMV virus was not a prerequisite for this elicitation and isolated coat protein (CP) subunits could also elicit the fast oxidative burst. The rapid induction of ROS was prevented by both inhibitors of plant signal transduction and inhibitors of NAD(P)H oxidases, suggesting activation of a multi-step signal transduction pathway. Induction of intracellular ROS by TMV was detected in TMV-resistant and -susceptible tobacco cultivars isogenic for the N allele. The burst was also detected with strains of virus that either elicit (ToMV) or fail to elicit (TMV U1) N' gene-mediated responses. Hence, early ROS generation is independent or upstream of known genetic systems in tobacco that can mediate hypersensitive responses. Analysis of other viruses and TMV CP mutants showed marked differences in their ability to induce ROS showing specificity of the response. Thus, initial TMV-plant cell interactions that lead to early ROS induction occur outside the plasma membrane in an event requiring specific CP epitopes.

Capsid↗
PubMed1990

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.

Capsid↗
PubMed1995

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.

Cloning, Molecular↗
PubMed1997

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.

Australia↗
PubMed2001

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

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

Genes, Viral↗
PubMed1975

Tracheal mucous transport in experimental canine asthma.

Tracheal mucous velocity (TMV) was measured in experimental canine asthma. All 10 sensitive dogs exposed to an aerosol of Ascaris suum extract showed a decrease in TMV to about one-third of base-line values within 45 min whereas only half of them (reactors) responded with bronchospasm as measured by a significant reduction in specific respiratory system conductance (SGrs). Mean TMV remained decreased at the end of the 2-h observation period when mean SGrs had returned to base-line values in the reactors. Neither TMV nor SGrs changed in nonsensitive animals who inhaled ragweed antigen. Aerosols of acetylcholine and histamine increased TMV thereby excluding these mediators as responsible for the impairment of mucous transport in asthma. Nebulization of A. suum antigen together with a selective antagonist of slow reacting substance of anaphylaxis (srs-a) produced a marked increase of TMV in all dogs regardless of whether or not bronchospasm was elicited. This suggests that the observed decrease in TMV may be related to the release of SRS-A during the immunologic reaction.

Animals↗
PubMed1983

Duration of mucociliary dysfunction following antigen challenge.

Antigen-induced bronchospasm is associated with an impairment of airway mucociliary clearance, but the duration of this defect has not been examined. In the present study, we determined the time course of mucociliary dysfunction following inhalation challenge with Ascaris suum extract in conscious sheep with hypersensitivity to this antigen. Tracheal mucous velocity (TMV) as an index of mucociliary function, pulmonary resistance (RL), and arterial oxygen tension (Pao2) were measured prior to, immediately after, and serially for 8 h after inhalation challenge. TMV and Pao2 were then measured periodically for at least 2 wk. Immediately after A. suum challenge, mean RL increased to 264% of base line (P less than 0.05), and mean Pao2 decreased to 61% of base line (P less than 0.05). By 2 h postchallenge, mean RL and Pao2 had returned to base line but mean TMV had fallen to 61% of base line (P less than 0.05). Mean TMV reached a minimum of 35% of base line after 5 h (P less than 0.05) and remained significantly depressed between 37% and 54% of base line for 7 days; by day 9, mean TMV had returned to base line. Inhalation challenge with a control antigen (ragweed) failed to alter mean TMV, RL, and Pao2 during the first 8 h and TMV and Pao2 at any time throughout the 2-wk observation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Abnormal mucociliary transport in allergic patients with antigen-induced bronchospasm: role of slow reacting substance of anaphylaxis.

We evaluated the possible role of slow reacting substance of anaphylaxis (SRS-A) in mediating mucociliary dysfunction in allergic asthma. In 6 asymptomatic nonsmokers with ragweed asthma, we measured specific airway conductance (SGaw) and tracheal mucous velocity (TMV) before and after bronchial challenge with ragweed extract, with or without pretreatment with 0.5% and 1% FPL-55712 (SRS-A antagonist). Mean baseline TMV was 8.9 mm/min (SD, 1.1). Placebo and FPL-55712 per se had no effect on TMV. With placebo pretreatment, the doses of ragweed extract that resulted in a decrease in SGaw by more than 35% from baseline, led to an immediate decrease in TMV to 74% of baseline (p less than 0.05), returning to baseline within 2 h. With 0.5% and 1% FPL-55712 pretreatment, doses of ragweed extract that resulted in a similar decrease in SGaw led to an increase in TMV to 130% and 126% of baseline (p less than 0.05), respectively, immediately after antigen challenge and returned to baseline 2 h postchallenge. Inhalation of 1% FPL-55712 immediately after antigen challenge prevented the decrease in TMV. These results indicate that (a) SRS-A liberated during airway anaphylaxis impairs mucous transport, and (b) the antigen-induced increase in TMV after pretreatment with an SRS-A antagonist may reflect a stimulatory effect of other chemical mediators of anaphylaxis.

Adolescent↗
PubMed1975

Appearances of microtubules after various fixative procedures, and comparison with the appearances of tobacco mosaic virus.

Microtubules in crane-fly spermatids appeared altered when the glutaraldehyde-fixed cells were not postfixed with osmium tetroxide. The cytoplasmic microtubules were altered more than the doublet microtubules. Addition of osmium tetroxide after dehydration did not produce appearances identical with those of microtubules postfixed directly after glutaraldehyde, and thus at least some alterations occurred during dehydration, possibly due to extraction of microtubule-associated lipid. The omission of osmium tetroxide postfixation did not cause drastic alterations in the appearances of either tobacco mosaic virus (TMV), or polymerized tobacco mosaic virus protein (without RNA), suggesting that microtubule stability is different from TMV stability (with respect to the embedment procedure). The electron-dense stain associated with embedded-sectioned TMV is predominantly outside the TMV protein, as demonstrated by the known distribution of TMV protein compared with the dimensions of sectioned TMV and negatively stained TMV. The same might hold true for microtubules, as evidenced by the dimensions of negatively stained, isolated brain microtubules compared with those of embedded and sectioned brain microtubules.

Acetates↗
PubMedDate not supplied

The discovery of the chemical nature of tobacco mosaic virus.

The path to arrive at the elucidation of the chemical nature of plant viruses was greatly facilitated by the availability of Tobacco mosaic virus (TMV) as biological tool. The first hypothesis on the chemical nature of TMV was advanced in 1899 by the American Albert Wood, who suggested an enzyme nature. This hypothesis, severely questioned by Harry Hallard in 1915, was re-proposed by several virologists. In 1926, the American Maurice Mulvania concluded that the virus might be a protein with the biological characteristics of an autocatalytic enzyme. Before arriving at the experimental evidence it was necessary to resolve two questions: the estimation of virus infectivity in quantitative terms, performed by Francis Holmes in 1928, and the purification of the virus, performed by Carl George Vinson between 1927 and 1934. Vinson gave a conclusive contribution to solve the question of the chemical nature of TMV by settling the protocol of TMV purification. He put forward the hypothesis of the protein nature in the early 1930s but had not the required firm belief to gave the final experimental evidence of it. Who first arrived at the experimental evidence of the protein nature of the virus was the American Wendell Meredith Stanley, in 1935. His celebrated work, a classic of the fundamental Virology, was followed by several papers in which this result was firmly reaffirmed. The heuristic value of Stanley's discovery held out a year: the decisive evidence of the actual chemical nature of TMV was offered in the late 1936 by an English group under the leadership of Frederick Charles Bawden. In their short paper, Bawden and co-operators demonstrated that TMV had a ribonucleoprotein nature, a result that was confirmed in the following years for several TMV strains and other viruses. Stanley and his group did accept this result only after a year of reticence and contradictions. The conversion to the ribonucleoprotein nature raised a dignified protest by Bawden and the sarcasm of his closest co-operator, Norman Wingate Pirie, because Stanley proved to be very reluctant to recognize the merit of the English group. The world of Virology continues to consider Stanley as the first scientist who elucidated the actual nature of a virus, and this eminent scientist was awarded the Nobel Prize for Chemistry, in 1946. By examining the papers Stanley published from 1937 to 1945, one can however find proof of his ambiguity, a fact that justifies the bitterness of Bawden and the sarcastic comments of Pirie.

History, 20th Century↗
PubMed2005

Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants.

When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.

Capsid Proteins↗
PubMedDate not supplied

[Effect of tobacco mosaic virus strains on the ultrastructure of parenchymal tobacco leaf cells].

It is shown that considerable amount of virus particles accumulates in the parenchymal cells infected with any of strains of tobacco mosaic virus (TMV) isolated from affected plants of papper (TMV-p), tomato (TMV-t), eggplant (TMV-e). Along with normal, abnormal (swolled and "thin") virions were found that testifies to their destruction. In reply to infection, activation of the lysosomal compartment was observed in the cells that expressed in the formation of cytoplasmic vacuoles, dyctiosomes, smooth endoplasmic reticulum elements, different vesicles, mitochondria with invaginations, multivesicular bodies. It is established that the investigating strains, in dependence on degree of stimulation of lysosomal compartment and development of intracellular lytic processes causing the destruction of virus particles and cellular structures, may be arranged as follows: TMV-p, TMV-e, TMV-t.

Cell Nucleus↗
PubMed1977

Effect of amino acid replacement on the stability of the tobacco mosaic virus protein structure.

A comparative polarographic study on the alkaline degradation of tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483, having histidine instead of glutamine at position 9 in the polypeptide chain, was performed. In the course of alkaline degradation and subsequent incubation in the supporting electrolyte at 0 degrees C TMV 483, unlike TMV vulgare, showed a polarographic effect indicating the unfolding of the TMV polypeptide. It was concluded that the replacement of glutamine-9 by histidine causes a decrease in the stability of the three-dimensional structure of the TMV protein subunit. A polarographic study of untreated virions as well as denatured proteins of both TMV strains showed that histidine, when incorporated into the polypeptide chain, is not active polarographically at the conditions used.

Glutamine↗
PubMedDate not supplied

[Virus-specific informosomes in tobacco cells infected with tobacco mosaic virus].

We have previously detected in TMV-infected cells virus-specific informosome-like ribonucleoproteins (vRNP) that differed in the CsCl buoyant density from mature TMV particles. It is shown in the present work that [3H]uridine-labelled TMV-specific structures, when fractionated in Cs2SO4, produce three types of structures, i. e. with a buoyant density of 1.23 g/cm3 (the so-called 1.23 material), 1.29 g/gm3 (mature virus) and 1.34--1.49 g/cm3 (vRNP). The 1.23 material has been investigated. The incorporation of [3H]palmitic acid and the sensitivity of this material to 0.1% Na dodecyl sulphate was interpreted to mean the presence of membrane components. Treatment of the 1.23 material Na dodecyl sulphate induces the release of the mature virus, vRNP and free viral RNA. vRNP was shown to contain genome TMV RNA (mol. weight, 2.0 x 10(6)) and a considerable amount of subgenomic TMV RNA (mol. weight, 1.1--1.3 x 10(6) and 0.6--0.8 x 10(6)). It is demonstrated that RNA isolated from vRNP codes for TMV-specific proteins and is able to hybridize with recombinant plasmid containing DNA-copy of 3'-end RNA TMV fragment (about one half of genome).

Centrifugation, Density Gradient↗
PubMed2007

Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins.

Hairy roots were used as an in vitro culture system for the propagation of wild-type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1-2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1-2 mg g(-1) dry weight or 20-28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV-based viral vector developed to express green fluorescent protein (GFP). This vector was about 260-fold less infectious than wild-type TMV and accumulated much more slowly in the roots. Maximum levels of TMV-GFP in the biomass were about 65-fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild-type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control.

Biomass↗
PubMed1994

Influence of heterologous tobamovirus movement protein and chimeric-movement protein genes on cell-to-cell and long-distance movement.

Sunn-hemp mosaic tobamovirus (SHMV) moves slowly from cell to cell in Nicotiana tabacum cv. Xanthi, but fails to move long distance. To determine the role of the SHMV movement protein (MP) in cell-to-cell and long-distance movement in tobacco, the SHMV MP gene was inserted into a TMV-cDNA clone that had approximately the 5'-half of the endogenous MP gene deleted. RNA transcripts inoculated onto tobacco induced systemic infections by 8 days postinoculation. Sequence analysis of the MP genes from purified virus isolated from systemically infected leaf tissue indicated that chimeric SHMV/TMV MP genes had been generated through RNA-RNA recombination within the 3'-termini of the MP gene sequences. When exchanged for the MP gene of TMV, three of four chimeric MP genes analyzed provided long-distance movement function for the hybrid viruses in tobacco. Two of the three hybrid viruses that moved long distance showed enhanced cell-to-cell movement relative to a recombinant TMV that expressed the intact SHMV MP gene. These observations suggest that the C-terminus of the TMV MP contains a determinant that can influence cell-to-cell movement in tobacco. A recombinant virus, TLSM, that expressed the intact SHMV MP gene exhibited cell-to-cell movement that was intermediate to SHMV and TMV, but failed to produce coat protein and was defective in long-distance movement. To further examine the role of the SHMV MP gene in long-distance movement, transgenic N. tabacum cv. Xanthi that expressed the wild-type SHMV MP gene were generated and found to facilitate rapid and efficient long-distance movement of a TMV mutant that contained a dysfunctional MP gene. Therefore, the inability of SHMV to systemically infect tobacco is a function of virus components and sequences other than those encoded by the SHMV MP gene.

Base Sequence↗
PubMed1995

Tobamovirus and dianthovirus movement proteins are functionally homologous.

The movement proteins (MPs) of tobacco mosaic tobamovirus (TMV) and red clover necrotic mosaic dianthovirus (RCNMV) enlarge plasmodesmata size exclusion limits, transport RNA from cell to cell, and bind nucleic acids in vitro. Despite these functional similarities, they have no sequence homology. However, they do appear to have similar secondary structures. We have used transgenic plants expressing either the TMV MP or the RCNMV MP, and a chimeric TMV that encodes the RCNMV MP as its only functional MP gene, to demonstrate that the MPs of TMV and RCNMV are functionally homologous. Further, both TMV and RCNMV can act as helper viruses to allow the cell-to-cell movement of the heterologous movement-defective viruses. These data support the conclusion that, despite other differences, such as particle morphology, host range, and sequence, TMV and RCNMV share a common mechanism for cell-to-cell movement.

Blotting, Northern↗