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PubMed1993

Analysis of regulatory elements involved in stress-induced and organ-specific expression of tobacco acidic and basic beta-1,3-glucanase genes.

Infection of tobacco by tobacco mosaic virus (TMV) induces coordinate expression of genes encoding acidic and basic beta-1,3-glucanase isoforms. These genes are differentially expressed in response to other treatments. Salicylate treatment induces acidic glucanase mRNA to a higher level than basic glucanase mRNA. Ethylene treatment and wounding strongly induce the basic glucanase genes but have little effect on genes encoding the acidic isoforms. Furthermore, the basic glucanase genes are constitutively expressed in roots and lower leaves of healthy plants, whereas the acidic glucanase genes are not. In order to investigate how these expression patterns are established, we fused promoter regions of an acidic and a basic glucanase gene to the beta-glucuronidase (GUS) reporter gene and examined expression of these constructs in transgenic tobacco plants. A fragment of 1750 bp and two 5'-truncated fragments of 650 bp and 300 bp of the acidic glucanase promoter were tested for induction of GUS gene expression after salicylate treatment and TMV infection. Upstream sequences of 1750 bp and 650 bp were sufficient for induction of the reporter gene by salicylate treatment and TMV infection, but the activity of the 300 bp fragment was strongly reduced. The results suggest that the 1750 bp upstream sequence of the acidic glucanase gene contains multiple regulatory elements. For the basic glucanase promoter it is shown that 1476 bp of upstream sequences were able to drive expression in response to TMV infection and ethylene treatment, but no response was found to incision wounding. Furthermore, high GUS activity was found in lower leaves and roots of healthy transgenic plants, carrying the 1476 bp basic glucanase promoter/GUS construct. When the promoter was truncated up to position -446 all activity was lost, indicating that the region between -1476 and -446 of the basic glucanase promoter is necessary for organ-specific and developmentally regulated expression as well as for induced expression in response to infection and other stress treatments.

Base Sequence↗

PubMed1988

Expression of tobacco mosaic virus RNA in transgenic plants.

Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in plants.

Base Sequence↗

PubMed2003

Gas chromatography-mass spectrometry with solid-phase microextraction method for determination of methyl salicylate and other volatile compounds in leaves of Lycopersicon esculentum.

Methyl salicylate (MeSA) in many plants is a important signaling compound, which plays an important role in a pathogen-induced defense response. In this paper, gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME) was developed for determination of MeSA and other volatile compounds in leaves of a tomato plant (Lycopersicon esculentum). Tomato leaves were ground under liquid nitrogen and sampled by HS-SPME, with a 100 microm polydimethylsiloxane fiber, and finally analyzed by GC-MS. Eighteen compounds in the leaves of tomato plant infested by tobacco mosaic virus (TMV) were separated and identified, among them MeSA, which was quantitatively analyzed by the standard addition method. MeSA concentrations higher than 2.0 microg g(-1) fresh weight accumulated in leaves of TMV-infested tomato plant as the defense response to TMV. A similar concentration of MeSA in the leaves of MeSA-treated tomato plant was also found. No MeSA in leaves of control tomato plant was detected. These findings suggest that MeSA might be a signaling compound in the tomato plant response to TMV. The present method for determination of MeSA required only simple sample preparation and no organic solvent, and provided an excellent relative standard deviation of less than 5.0% and a low detection limit of 10 ng g(-1) fresh weight for MeSA. These results show that GC-MS-HS-SPME is a simple, rapid and sensitive method for determination of MeSA and other plant-signaling compounds in plant tissues.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum.

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P(0). Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P(0) and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK(c)1) gene was increased specifically in the incompatible interaction with TMV-P(0). The expression of CaPK(c)1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK(c)1 responds to several defense-related abiotic stresses in addition to TMV infection.

Amino Acid Sequence↗

PubMed1998

Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses.

The 3' terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3' non-coding region of 235 nucleotides, coat protein (CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages.

Amino Acid Sequence↗

PubMed2005

Development of a quantitative tool for measuring changes in the coefficient of conductivity of plasmodesmata induced by developmental, biotic, and abiotic signals.

The regulation of intercellular and interorgan communication is pivotal for cell fate decisions in plant development and probably plays a significant role in the systemic regulation of gene expression and in defense reactions against pathogens or other biotic and abiotic environmental factors. In plants, symplasmic cell-to-cell communication is provided by plasmodesmata (Pd), coaxial membranous tunnels that span cell walls interconnecting adjacent cytoplasms. Macromolecules, proteins, and RNA may be transported through Pd by passive diffusion or by a facilitated mechanism. A quantitative tool was developed to measure the coefficient of conductivity, C(Pd), for diffusion-driven transport via Pd and to assess changes in the coefficient induced by developmental, biotic and abiotic signals. (GFP)C(Pd), the coefficient of conductivity for cell-to-cell spread of green-fluorescent protein (GFP), a protein with a Stokes radius of 2.82 nm, was determined in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana plants expressing the movement protein of tobacco mosaic virus (MP(TMV)) incubated both in dark and light and at 16 and 25 degrees C. Under all conditions, Pd in source leaves conducted macromolecules, with (GFP)C(Pd)sink>(GFP)C(Pd)source. Light down-regulated (GFP)C(Pd) (all conditions); down-regulation was stronger for sink cells. The effect of MP(TMV) on (GFP)C(Pd) between epidermal cells was dependent on temperature and leaf development; at 16 degrees C, MP(TMV) down-regulated (GFP)C(Pd) only in source leaves, while at 25 degrees C, MP(TMV) had no significant effect. This quantitative tool should be useful for investigating differences in Pd conductivity that are induced by mutations or silencing.

Biological Transport, Active↗

PubMed2005

Tobacco ZFT1, a transcriptional repressor with a Cys2/His2 type zinc finger motif that functions in spermine-signaling pathway.

We previously proposed that a spermine (Spm)-mediated signal transduction pathway is involved in the hypersensitive response induced by Tobacco mosaic virus (TMV) in tobacco plants. To identify regulatory component(s) of this pathway, we surveyed a tobacco cDNA library and found that the ZFT1 gene, which encodes a Cys2/His2 type zinc-finger protein, is Spm-responsive. ZFT1 was not induced by two other polyamines, putrescine and spermidine, or by salicylic acid (SA), jasmonic acid or ethylene. Furthermore, ZFT1 was upregulated in TMV- inoculated tobacco plants in an N gene-dependent manner. Notably, induction of ZFT1 by Spm and by TMV infection was unimpaired in NahG-transgenic tobacco plants, indicating that cross-talk with an SA signaling pathway is not involved in this response. Within the Spm-signaling pathway, we found that ZFT1 functioned downstream of both mitochondrial dysfunction and mitogen-activated protein kinase activation. The ZFT1 protein has two zinc finger motifs and shows a high degree of similarity to ZPT2-3 in petunia and SCOF1 in soybean. However, unlike the latter two proteins, ZFT1 binds to the EP1S sequence and functions as a transcription repressor. Moreover, interestingly, ZFT1 overexpression rendered tobacco plants more tolerant to TMV. Based on the results presented here, we propose that ZFT1 functions as a transcription repressor in a Spm signaling pathway, thereby accelerating necrotic local region formation in tobacco leaves.

Amino Acid Sequence↗

PubMed2006

Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a tobacco mosaic virus vector through agroinfection.

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus-host plant systems.

Base Sequence↗

PubMed1976

The rye embryo system as an alternative to the wheat system for protein synthesis in vitro.

Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.

Cell Fractionation↗

PubMed1995

Systemic production of foreign peptides on the particle surface of tobacco mosaic virus.

By using a new tobacco mosaic virus (TMV) vector [Hamamoto, H., et al. (1993) Bio/Technology 11, 930-932], we have constructed TMV particles which present three different kinds of epitopes, two of them from influenza virus hemagglutinin (HA), and one from human immunodeficiency virus type I (HIV-I) envelope protein, on the surface of the particles. Each of these TMV particles reacted with each anti-peptide antiserum. These results suggest that this TMV vector can be used as an antigen presentation system.

Amino Acid Sequence↗

PubMed1988

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.

Base Sequence↗

PubMed1983

Tobacco mosaic virus protein synthesis is correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis.

The synthesis rates of three proteins of tobacco mosaic virus (TMV), 160, 110, and 17.5 kDa, were monitored at intervals after interruption of synthesis of TMV RNA. Following inhibition of synthesis of both single-stranded and double-stranded RNAs by shifting wild type TMV to 40 degrees or ts mutant III2-35 to 35 degrees, the synthesis rates of viral proteins declined sequentially, with that of the larger proteins declining faster. When viral RNA synthesis was prevented with cordycepin, synthesis rates of the 110 and 160-kDa proteins declined rapidly, while the 17.5-kDa protein decreased more slowly. These data imply that the functional mRNA is transitory, probably nascent RNA, and that each protein is produced independently. The process of translation of viral mRNA was not temperature sensitive and occurred normally for brief periods after shift to restrictive temperatures. When single-stranded RNA synthesis was inhibited differentially from double-stranded RNA synthesis, protein synthesis was correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis. Following a shift of ts mutant IV-35 to 35 degrees, a shift that immediately stopped single-stranded RNA synthesis without inhibiting double-stranded RNA synthesis, all three viral proteins continued to be produced normally. Also, after return of wild type TMV to 25 degrees after a 1-hr incubation at 40 degrees, viral protein and double-stranded RNA synthesis recovered in parallel to the normal rate after 8 hr whereas single-stranded RNA synthesis, which had been reduced more drastically, recovered more slowly after 16 hr.

Deoxyadenosines↗

PubMed1986

Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-biphosphate carboxylase share a common antigenic determinant.

An immunological relationship was detected between the coat protein of the common (U1) strain of tobacco mosaic virus (TMV) and the large subunit of the ubiquitous CO2-fixing host enzyme, ribulose-1,5-biphosphate carboxylase (RuBisCo). When assayed by Western immunoblotting or indirect ELISA, polyclonal antisera to TMV coat protein and to RuBisCo reacted with both antigens. In addition, a monoclonal antibody specific for the C-terminal antigenic determinant of TMV coat protein reacted with RuBisCo. Conversely, several monoclonal antibodies generated to the large subunit of RuBisCo reacted with TMV coat protein. This cross-reactivity was verified by an examination of the amino acid sequences of both proteins. A region of homology was found between the carboxy proximal portion of coat protein and the sequence 60-73 residues from the amino terminus of RuBisCo large subunit. This homology was not mirrored at the nucleic acid level because of different codon usages for the two proteins.

Amino Acid Sequence↗

PubMed1989

Effect of protein aggregation state on coat protein-mediated protection against tobacco mosaic virus using a transient protoplast assay.

To address the mechanism(s) of protection against tobacco mosaic virus (TMV) infection conferred by expression of the TMV capsid protein (CP) gene in transgenic tobacco plants, a transient protection assay has been developed. Introduction of either purified viral CP or virus inactivated by ultraviolet irradiation into tobacco protoplasts induced a transient protection to challenge virus introduced concomitantly or shortly thereafter. The transient protection was characterized and the effects of different aggregation states of TMV CP were tested in the transient assay system. Tobacco mosaic virus CP preparations composed largely of helical, virus-like, aggregates conferred a less transient protection against TMV and greater protection against a distantly related virus than did preparations composed primarily of smaller aggregates.

Capsid↗

PubMed1990

Inhibition of uncoating of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene.

The uncoating of tobacco mosaic virus (TMV) particles in protoplasts isolated from leaves of transgenic tobacco plants that express the TMV coat protein gene was investigated. Extracts of these protoplasts collected up to 1 hr after inoculation with TMV contained fewer of the complexes ("striposomes") thought to be involved in cotranslational disassembly of virus particles than did extracts of protoplasts that do not express the viral coat protein gene. These results are consistent with the hypothesis that TMV coat protein-mediated resistance is at least in part the result of inhibition of the uncoating of the virus particles in the inoculum.

Capsid↗

PubMed1991

Altered function of the tobacco mosaic virus movement protein in a hypersensitive host.

The N gene in Nicotiana sp. confers hypersensitive resistance to all strains of tobacco mosaic virus (TMV) and limits the rate of virus spread in infected leaves. To examine the role of the movement protein (MP) of TMV in the hypersensitive reaction (HR), transgenic Nicotiana tabacum cv. Xanthi-nc (genotype NN) plants that express the MP gene were produced and the molecular size exclusion limit of plasmodesmata in leaf mesophyll cells was monitored. At the HR-permissive temperature (24 degrees) movement from cell to cell of fluorescein isothiocyanate-labeled dextran of molecular mass 3.9 kDa was detected while 9.4-kDa molecules failed to move. At the HR-nonpermissive temperature (33 degrees) the 9.4-kDa probe moved readily from cell to cell. In contrast, in transgenic Xanthi (genotype nn) which express the MP gene the 9.4-kDa probe moved from cell to cell at 24 and 33 degrees. These results suggest that the N gene may modify the ability of the MP to alter plasmodesmata molecular exclusion limits, although expression of the TMV-MP gene alone did not induce the HR. Furthermore, when MP(+) Xanthi-nc tobacco lines were inoculated with a TMV that lacked a MP gene the HR was induced, and the concentration of MP in the transgenic lines was correlated with the degree of the HR.

Capsid Proteins↗

PubMed1991

In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants.

A full-length cDNA clone of the U1 (common) strain of tobacco mosaic virus (TMV) was constructed, and highly infectious transcripts were produced in vitro using bacteriophage T7 RNA polymerase. Frameshift mutations designed to cause premature termination of translation were introduced into either the 30-kDa movement protein (MP) gene or the coat protein (CP) gene. The MP-frameshift mutant was unable to locally or systemically infect inoculated tobacco plants. However, inoculation of transgenic tobacco plants that expressed a wild-type TMV MP gene resulted in both local and systemic viral infection. The CP-frameshift mutant, although unable to move systemically in nontransformed tobacco, exhibited systemic movement in transgenic plants that expressed a wild-type TMV CP gene. Transgenic tobacco plants that expressed the appropriate wild-type TMV gene were thus able to complement, in trans, mutant viruses lacking a functional MP or CP gene.

Amino Acid Sequence↗

PubMed1991

Mutation and replacement of the 16-kDa protein gene in RNA-1 of tobacco rattle virus.

The function of the 16-kDa protein encoded by tobacco rattle virus (TRV) RNA-1 was investigated by a mutational analysis of the 16-kDa protein gene. Transcripts of TRV RNA-1 produced from a full-length cDNA clone of TRV RNA-1 (SYM strain) remained infectious when the 16-kDa protein gene was disrupted by premature termination codons and a deletion which removed 73% of the coding region. A deletion which included the intergenic region between the 29-kDa protein gene and the 16-kDa protein gene, the entire 16-kDa protein coding region, and 57% of the 3' noncoding region was not infectious. Transcripts in which the 16-kDa protein coding region was replaced by the tobacco mosaic virus (TMV) (L strain) coat protein gene were also infectious and expressed TMV coat protein in infected tissue. Inclusion of the TMV origin of assembly sequence in the chimaeric constructs resulted in the accumulation of TMV-like virus particles in infected tissue.

Blotting, Northern↗