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PubMed1997

Resistance to tobamoviruses in transgenic tobacco plants expressing the coat protein gene of pepper mild mottle virus (Korean isolate).

Red pepper, one of the most important vegetable crops in Korea, is severely affected by viral diseases causing 20-50% reduction in product yield. A pepper strain of tobacco mosaic virus (TMV-p) is the most common virus in red pepper. To study the molecular structure of the TMV-p virus, we generated cDNA clones of the viral genome. Partial sequencing of a few cDNA clones revealed that TMV-p shares a 98% identity at the nucleotide level with the Spanish isolate of pepper mild mottle virus (PMMV-s). This suggests that TMV-p should be reclassified as the Korean isolate of PMMV (PMMV-k). The coat protein (CP) gene together with the 3' untranslated region of the PMMV-k virus was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligomers deduced from the sequence of PMMV-s. The sequence analysis of the CP gene and the 3' untranslated region further confirmed that PMMV-k is highly related to PMMV-s. The CP gene and the 3' untranslated region of PMMV-k were cloned into a plant expression vector and the construct was introduced into tobacco plants. The transgenic plants expressing the PMMV-k CP gene were delayed in developing systemic disease or failed to develop symptoms at all after inoculation with PMMV-k. Delay of symptoms was also observed when the plants were inoculated with TMV-OM which shares a 74% homology with PMMV-k in the amino acid sequence of the CP region. In a local lesion host, the CP expressing plants exhibited a greatly reduced number of necrotic lesions as compared to control plants after inoculation with TMV-OM. Our results show that CP-mediated viral resistance is readily applicable in the case of PMMV-k and can provide resistance to other viruses in the tobamovirus group.

Amino Acid Sequence↗

PubMed1987

Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature-sensitive mutant defective in cell-to-cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one-base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame-shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame-shift mutants were replication-competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell-to-cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis-acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.

Journal Article↗

PubMedDate not supplied

The effect of systemic hydration on normal and impaired mucociliary function.

Whether systemic hydration enhances the clearance of lower airway secretions in normal lungs and in lungs during antigen-induced bronchial obstruction was studied. In five conscious allergic sheep, tracheal mucus velocity (TMV) as an index of lower airway mucociliary clearance was measured before and serially for 60 minutes following intravenous infusion of 5% dextrose solution at volumes ranging from 5 to 35 ml/kg. None of these fluid regimens had a significant effect on TMV, although there was a tendency toward a slowing of TMV at the higher volumes of 25 and 35 ml/kg. After producing an acute allergic airway response by inhalation challenge with specific antigen, there was a decrease in mean TMV by 27% and 15% before sham infusion and infusion of 35 ml/kg of dextrose solution, respectively. During the subsequent 60-minute observation period, mucociliary transport was further impaired by dextrose infusion, with mean TMV being lower after dextrose infusion than after sham infusion at all times of measurement (P less than 0.05). The lowest value was reached 30 minutes after dextrose infusion (45% of control). It was concluded that systemic hydration does not significantly alter airway mucociliary transport in the normal state but further impairs mucociliary transport during allergic mucociliary dysfunction. This raises questions regarding the therapeutic role of systemic hydration in the treatment of children and adults who have bronchial asthma.

Animals↗

PubMed1997

Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro.

It was found that the fusion (His)6-movement proteins (MPs) of two tobamoviruses (TMV UI and a crucifer-infecting tobamovirus, crTMV) were efficient nonspecific translational repressors. The in vitro translation of viral RNAs was blocked by incomplete 30K MP-RNA complexes formed at the MP:RNA molar ratios of 100-150:1. Similar results were obtained with the barley stripe mosaic hordeivirus (BSMV)-encoded 58K MP; however, the translation inhibiting activity of the 58K MP was manifested only in the presence of magnesium. By contrast, the 25K MP of potato virus X (PVX) was incapable of forming MP-RNA complexes under experimental conditions used and did not inhibit in vitro translation. The translation repressing ability correlated with the level of MP affinity to RNA. The complexes of the 30K MP and 58K MP with TMV RNA were not infectious in isolated protoplasts; however, they were infectious in indicator plants. Reduction of MP affinity to RNA resulted in translatability of MP-TMV RNA complexes that apparently was due to their destabilization. Thus, the deletion mutant DEL4 MP formed MP-TMV RNA complexes that were translatable in vitro, infectious to protoplasts and plants. In contrast to this, the complexes of TMV RNA with the mammalian RNA-binding protein p50 were nontranslatable and noninfectious to either protoplasts or intact plants. These results implied that nontranslatable MP-RNA complexes which could not replicate in the primary infected cells were converted into a translatable and replicatable form in the course of passage through plasmodesmata in planta.

Collodion↗

PubMed1999

Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability.

Previously we showed that the ribonucleoprotein complexes (RNPs) of the TMV 30-kDa movement protein (MP) with TMV RNA are nontranslatable in vitro and noninfectious to protoplasts, but are infectious to intact plants. It has been suggested that MP-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata (Karpova et al., 1997, Virology 230, 11-21). The role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. Phosphorylation of the TMV MP, either before or after RNP complex formation, caused a conversion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.

Brassica↗

PubMed1993

Plant viral leaders influence expression of a reporter gene in tobacco.

In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter beta-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two- to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four- to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four- to six-fold more efficiently than the random leader control.

Base Sequence↗

PubMed1991

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding.

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus, ORSV-Cy, the Korean isolate.

The partial nucleotide sequence of the 3'-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3' end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).

Amino Acid Sequence↗

PubMed1991

Isolation of mutants of Arabidopsis thaliana in which accumulation of tobacco mosaic virus coat protein is reduced to low levels.

We have found that Arabidopsis thaliana is susceptible to infection with a crucifer strain of tobacco mosaic virus (TMV-Cg); the coat protein of TMV-Cg accumulated to a high level in uninoculated rosette leaves several days after inoculation. As a first step in the search for host-coded factors that are involved in virus multiplication, we isolated mutants of A. thaliana in which the accumulation of TMV-Cg coat protein was reduced to low levels. Of 6000 M2 plants descended from ethyl methanesulfonate-treated seeds, two such lines (PD114 and PD378) were isolated. Genetic analyses suggested that the PD114 phenotype was caused by a single nuclear recessive mutation, and that PD114 and PD378 belonged to the same complementation group. The coat protein accumulation of a tomato strain of TMV (TMV-L) was also reduced in PD114 plants compared to that in the wild-type plants. In contrast, PD114 plants infected with turnip crinkle or turnip yellow mosaic viruses, which belong to taxonomic groups other than Tobamovirus, expressed similar levels of these coat proteins as did infected wild-type plants.

Capsid↗

PubMed1986

Immunological properties and biological function of monoclonal antibodies to tobacco mosaic virus.

Monoclonal antibodies to TMV vulgare produced in hybridoma cultures as well as in ascitic fluid were characterized according to their reactivity with the virion and/or the coat protein monomer thus revealing specificity for epitopes, cryptotopes or neotopes. Different patterns of crossreactivity of these monoclonal antibodies with TMV strains dahlemense and Holmes' Ribgrass occurred. Some monoclonal antibodies showed stronger reactivity with these strains than with the immunizing strain. The monoclonal antibodies were TMV-specific as they did not react with ArMV and PLRV and proteins of healthy tobacco plants. The monoclonal antibodies were of the IgG2a or IgM isotype. The specific activity (Ext405nm/hour/100 micrograms MCA) with the immunizing virus and its coat protein monomers was determined as characteristic property of each monoclonal antibody. A monoclonal antibody specific for the C-terminal epitope of TMV coat protein was selected by means of the corresponding chemically synthesized tetrapeptide. With this monoclonal antibody infectivity of TMV was neutralized.

Antibodies, Monoclonal↗

PubMed2005

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper.

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant's defense signal pathway against pathogens.

Alanine Transaminase↗

PubMed2000

Recombination between a 3-kilobase tobacco mosaic virus transgene and a homologous viral construct in the restoration of viral and nonviral genes.

Transgenic plants harboring various plant virus sequences have shown resistance to viral infections. An environmental risk associated with the use of these plants is the possibility of forming a novel virus by recombination between challenging viruses and transgenic viral mRNA. Two experiments were designed using tobacco mosaic virus (TMV) vectors and transgenic Nicotiana benthamiana to determine if recombinant viral RNA would be detectable. N. benthamiana was transformed with a nontranslatable portion of a TMV viral vector including part of the replicase gene, the movement protein gene, a gene for green fluorescent protein (GFP), and the coat protein gene. When transformed plants were inoculated with a TMV vector coat protein mutant which could not move efficiently through the host, recombinant RNA was detected in 32% of the infected plants, although virions were not detected. When transformed plants were infected with a TMV vector with a normal coat sequence but three base changes in the GFP sequence, no recombinant RNA or virions were detected. Thus, recombinant RNA between TMV RNA and host mRNA did not accumulate to detectable levels under nonselective conditions, and though recombinant RNA did accumulate in the presence of selective pressure, an encapsidated recombinant viral population did not develop.

Capsid↗

PubMed1987

Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins.

Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.

Antibody Specificity↗

PubMed1989

A tobacco mosaic virus-hybrid expresses and loses an added gene.

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.

Chloramphenicol O-Acetyltransferase↗

PubMed1989

Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis-related PR1b gene.

Correlation of the temporal and spacial pattern of induction of the pathogenesis-related (PR) genes PR1a, PR1b, and PR1c with viral infections in certain tobacco cultivars has implicated PR proteins in viral disease resistance. To test whether the PR1 proteins of tobacco are involved in viral resistance, transgenic Nicotiana tabacum plants were constructed which constitutively express the PR1b gene. This protein was secreted from cells of transgenic plants and accumulated in the extracellular space at levels equivalent to those found in nontransgenic plants in association with disease resistance. Transgenic plants derived from the cultivar (cv.) Xanthi (susceptible to tobacco mosaic virus [TMV] infection) exhibited no delayed onset or reduction in the severity of systemic symptoms after TMV infection. In transgenic plants derived from cv. Xanthi-nc (TMV resistant), the time of appearance, the size and general morphology, and the number of viral lesions produced were similar to the parental control plants after TMV infection. These data indicate that the PR1b protein of tobacco is not sufficient for TMV resistance, and imply that the PR1 proteins may not function as unique antiviral factors.

Cell Transformation, Viral↗

PubMed1987

Immunochemical studies of tobacco mosaic virus--VII. Use of comparative surface accessibility of residues in antigenically related viruses for delineating epitopes recognized by monoclonal antibodies.

The binding properties of 18 monoclonal antibodies (MAbs) directed against the tobacco mosaic virus (TMV) coat protein were studied with five related tobamoviruses and seven mutant viruses, as well as with the dissociated coat proteins of these variants. Ten of the antibodies bound to both TMV and TMV protein, but these were able to discriminate between different mutants only when whole virus particles were compared in the immunoassay. Three MAbs reacted with TMV but not with dissociated viral subunits and these recognized the same residue exchanges. Five MAbs recognized synthetic peptides of 13-28 residues corresponding to parts of the protein. By comparing the common accessible surface between TMV and antigenically related viruses, it was possible to narrow down the region recognized by some of these MAbs. The linear peptide sequence recognized by these antibodies did not represent the entire epitope and residue exchanges around this region were able to affect the binding of MAbs.

Amino Acid Sequence↗

PubMed1994

High-resolution electron microscopy of biological specimens in cubic ice.

Images of two biological test specimens, catalase and TMV, were recorded in cubic ice, prepared by controlled devitrification at -130 degrees C and -75 degrees C or in vitrified buffer. Cubic ice provides a rigid support for biological specimens which is stable under the electron beam to within about 1 A, as shown by images of the ice lattice. Neither catalase nor TMV were disrupted by the crystallization of vitrified water. Electron diffraction patterns of highly oriented rafts of TMV extending to 2.3 A resolution were used to judge the quality of TMV images. Structural high-resolution details of TMV was better in cubic ice, and the success rate for recording good images was higher than in vitrified medium.

Catalase↗

PubMed1994

The role of the 3'-untranslated region of non-polyadenylated plant viral mRNAs in regulating translational efficiency.

Tobacco mosaic virus (TMV) is a positive-sense RNA virus in which the single genomic RNA functions as a messenger RNA. It is a member of a class of plant viral RNAs that are the only known non-polyadenylated mRNAs in plants. The 3'-untranslated region (UTR) of TMV genomic RNA is the functional equivalent of a poly(A) tail in that it increases mRNA stability and regulates translational efficiency. To determine whether the 3'-UTR of other non-polyadenylated plant viral mRNAs regulate translation, those from turnip yellow mosaic (TYMV), brome mosaic (BMV), and alfalfa mosaic (AlMV) viruses were investigated. Chimeric gene constructs were made in which the viral 3'-UTRs were introduced immediately downstream from the reporter genes encoding beta-glucuronidase (GUS) and luciferase (LUC), and were translated in plant protoplasts following delivery of the mRNA using electroporation. The 3'-UTR from BMV RNA3 regulated reporter gene expression in vivo to an extent comparable to that observed for the TMV 3'-UTR. The BMV 3'-UTR increased both message stability and translational efficiency. As regulators of translation, the BMV and TMV 3'-UTR were dependent on the presence of a cap at the 5' terminus for function. The 3' UTR of TYMV or AlMV RNA4 had little impact on translation or transcript stability. These data suggest that although the TMV 3'-UTR is not unique in regulating translation, the 3'-UTR of plant viral mRNAs do vary in their regulatory ability.

Base Sequence↗