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At least 19 recordsLinked to original sources
PubMed1992

Production of the tobacco mosaic virus (TMV) transport protein in transgenic plants is essential but insufficient for complementing foreign virus transport: a need for the full-length TMV genome or some other TMV-encoded product.

We have reported previously that tobamoviruses enable the transport of red clover mottle comovirus (RCMV) in tobacco plants normally resistant to RCMV. Here we show that RCMV transport does not take place in transgenic tobacco plants (line To-4) producing the 30K transport protein of tobacco mosaic virus (TMV), whereas the transport of the TMV Ls1 mutant, the cell-to-cell movement of which is temperature sensitive, is complemented in these plants. However, RCMV transport is observed when these transgenic plants are infected with both RCMV and TMV Ls1 at the non-permissive temperature (33 degrees C). It is suggested that (i) the hypothetical modification of transgenic plant plasmodesmata by the TMV 30K transport protein can specifically mediate the cell-to-cell movement of the homologous virus (TMV), but is insufficient to mediate RCMV transport; (ii) the presence of the full-length TMV genome or a certain TMV-encoded product(s) besides the 30K protein is essential for complementation of the RCMV transport function. The possibility that line To-4 might provide enough 30K protein to complement TMV Ls1 but not RCMV cannot be ruled out. During double infection the mutant 30K protein may, in concert with the wild-type 30K protein, provide the transport function for RCMV.

Biological Transport↗
PubMed1989

Decreased levels of TMV coat protein in transgenic tobacco plants at elevated temperatures reduce resistance to TMV infection.

Transgenic tobacco plants that accumulate tobacco mosaic virus (TMV) coat protein (CP) are resistant to TMV infection under standard growth conditions. The amount of CP accumulated and the degree of resistance to TMV were found to be temperature dependent. Exposure to continuous high temperatures (30-35 degrees) results in a sharp decrease in the amount of CP within 6 hr with no further change for at least 6 days. Under these conditions the transgenic plants developed typical systemic disease symptoms when inoculated with TMV although disease development was delayed. Transgenic plants which were moved from 35 to 22 degrees accumulated the normal level of CP within several hours. Transgenic tobacco plants inoculated and held at 35/25 degrees day/night cycles retained resistance to TMV infection. The level of CP mRNA was constant at each temperature and was associated with polyribosomes. On the basis of these results we suggest that the low level of CP under elevated temperature is due to instability of the TMV CP. In contrast, TMV CP levels in transgenic tomato plants also dropped under elevated temperatures yet retained high resistance to TMV.

Blotting, Northern↗
PubMed1991

Replication of TMV-L and Lta1 RNAs and their recombinants in TMV-resistant Tm-1 tomato protoplasts.

Tm-1 is a gene that provides resistance to tomato plants against tobacco mosaic virus (TMV) infection. In tomato cells carrying the Tm-1 gene, multiplication of TMV is inhibited. From previous analysis of resistance-breaking mutants, the involvement of the 130- and 180-kDa proteins, putative viral replicases, in the resistance conferred by the Tm-1 gene was suggested. When wild-type TMV RNA was co-inoculated with a resistance-breaking mutant RNA, replication of the wild-type TMV genomic RNA could not be rescued by the 130- and 180-kDa proteins of a resistance-breaking strain, Lta1. To investigate how the putative resistance factor interacts with the 130- and 180-kDa proteins, we expressed the wild-type TMV protein sequence that is associated with the resistance-breaking phenomenon as part of a recombinant virus derived from Lta1 in Tm-1/Tm-1 protoplasts. No specific degradation of wild-type TMV protein sequences was observed, suggesting that the mechanism of the resistance does not involve the instability of a viral protein.

Amino Acid Sequence↗
PubMed1990

Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein.

Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) which express tobacco mosaic virus (TMV) U1 strain coat protein (CP) can complement both the assembly and the long-distance spread of CP-defective (DT1) or coat proteinless (DT1G) mutants of TMV. Both mutants arose spontaneously from PM2 and exist only as unencapsidated RNA in the inoculated leaves of control tobacco plants, where they are unable to form virus particles or to spread systemically. TMV CP expressed in transgenic tobacco plants [CP+ line 3404; P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738-743] was able to package some of either mutant viral RNA into TMV-like particles in vivo and resulted in the long-range spread of infection. In vivo encapsidated DT1 RNA was recovered and reinoculated onto control or new CP+ transgenic tobacco plants. Localized infection of control plants confirmed that no RNA recombination or reversion of the mutant RNA to wild-type had occurred during passage in the first CP+ plant. In contrast, encapsidated DT1 RNA was unable to produce even local infection in CP+ transgenic plants confirming that CP-mediated protection operates during the early stages of virus infection, including particle uncoating. By positive complementation, these results also confirm that TMV CP is required for the long-distance spread of infection.

Blotting, Northern↗
PubMed1979

Polar uncoating of tobacco mosaic virus (TMV) with dimethylsulfoxide (DMSO) and subsequent reassembly of partially stripped TMV.

Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3'end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3' end. Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5' direction. The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.

Dimethyl Sulfoxide↗
PubMed1989

The expression of the TMV-specific 30-kDa protein in tobacco protoplasts is strongly and selectively enhanced by actinomycin.

The TMV-encoded 30-kDa protein has been implicated in the cell-to-cell transport of TMV in the infected plant. The polyethylene glycol-mediated inoculation of tobacco protoplasts with TMV particles and TMV RNA was used to compare the time courses of the viral 30-kDa protein synthesis in vivo. Upon infection of protoplasts with TMV RNA, the synthesis of the viral 30-kDa protein starts after 4 to 6 hr, has its maximum after 8 to 10 hr, and decreases. After inoculation of protoplasts with TMV, however, the start of the viral 30-kDa protein synthesis and its maximum are delayed by 2 hr, followed by the same decrease. We show that actinomycin D dramatically stimulates the synthesis of the 30-kDa protein by up to 2 orders of magnitude, whereas the synthesis of the viral 126 kDa, the 183 kDa, and the coat protein is increased only by a factor of 2. Surprisingly, actinomycin V is twice as active as actinomycin D, whereas actinomycin I is nearly inactive. The specific stimulation of the 30-kDa synthesis by actinomycin D in vivo depends neither on the Nicotiana variety nor on the TMV strain used. Final evidence that the 30-kDa protein is truly TMV-derived is provided by the slightly different electrophoretic mobilities of the 30-kDa proteins encoded by TMV strains vulgare, dahlemense, and U2. The identification of the 30-kDa protein in two-dimensional gels was achieved for the first time by a combination of ionic and nonionic detergents for the solubilization of the 30-kDa protein and by the specific stimulation of its synthesis by actinomycin D. The mechanism of the strong and selective actinomycin effect on the viral 30-kDa protein synthesis in vivo is as yet obscure. Actinomycin does not appear to act directly on viral protein biosynthesis, since it neither stimulates the 30-kDa synthesis upon translation of TMV RNA in vitro nor alters the ratio of the products. Actinomycin may rather act by inhibiting selectively the synthesis of a host factor whose synthesis starts at least 4 hr after TMV infection and which strongly inhibits the expression of the viral 30-kDa transport protein.

Capsid Proteins↗
PubMed1988

Resistance to TMV in transgenic plants results from interference with an early event in infection.

Constitutive expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in transgenic tobacco plants results in inhibition of disease symptom development following inoculation with TMV. Evidence is presented here that this protection is also observed in leaf mesophyll protoplasts isolated from these plants. Protoplasts were resistant to infection by TMV at concentrations of 10 microgram/ml to 1 mg/ml when introduced by either electroporation or polyethylene glycol-mediated inoculation. There was little protection against infection by TMV RNA and the protection was lost as the concentration of TMV RNA in the inoculum increased. When virus was incubated briefly at pH 8.0 prior to inoculation, protection broke down in a manner similar to that observed following RNA inoculation. Analogous results were obtained in experiments with whole plants. Because virus treated in this manner has presumably lost little or no CP, these results suggest that expression of the TMV CP gene in transgenic plant cells prevents TMV from uncoating. A model is presented for the mechanism of this blockage which relates these results to early events in TMV infection.

Capsid↗
PubMed1988

Selective encapsidation of CAT gene transcripts in TMV-infected transgenic tobacco inhibits CAT synthesis.

Young tobacco seedlings (F1-progeny), transformed to express chloramphenicol acetyltransferase (CAT) mRNA with or without a 3'-proximal copy of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA (residues 5118-5550), were inoculated with TMV. After 21 days, virus symptoms were observed and systemic TMV infections were confirmed by Western blotting for viral coat protein and by electron microscopy of leaf saps. CAT activities were measured in extracts of leaf discs taken before, and 21 days after, virus inoculation. On average, the systemic leaves from TMV-infected CAT-transgenic plants containing the OAS exhibited 3.2-fold less CAT activity than the equivalent leaves from CAT-transgenic control plants lacking the OAS. Hence selective, OAS-dependent encapsidation of nuclear DNA transcripts into TMV-like (pseudovirus) particles can reduce expression of a particular mRNA, post-transcriptionally, in vivo. Furthermore, these data indicate that TMV self-assembly is not restricted to an exclusive subcellular compartment in vivo, and that formation of natural pseudovirions (A. Siegel, Virology 46, 50-59 (1971)) may shut off specific host RNA functions.

Acetyltransferases↗
PubMed1988

Selective recovery of foreign gene transcripts as virus-like particles in TMV-infected transgenic tobaccos.

A short origin-of-assembly sequence (OAS) located in the 30kDa movement protein gene, about 1.0kb from the 3'-end of the common strain of tobacco mosaic virus (TMV) RNA, nucleates encapsidation of the 6395-nucleotide-long genome by TMV coat protein in vitro, and presumably also in vivo. Single-stranded RNAs containing a foreign reporter gene sequence and the TMV OAS at their 5' - and 3' -ends, respectively, can be synthesized in vitro from recombinant SP6-transcription plasmids and will assemble spontaneously in vitro to form TMV-like 'pseudovirus' particles. In this paper, we show that foreign gene transcripts derived from the nuclear DNA of plants transformed by Agrobacterium tumefaciens, and which contain the TMV OAS, can be assembled into stable 'pseudovirus' particles in vivo during a systemic infection by TMV (helper). This is the first report of structural complementation between a heritable function bestowed on a transgenic plant and an infecting virus. As a route to protect, accumulate and recover a specific mRNA in vivo, in transgenic plant cells, this novel approach may find wider applications in developmental plant molecular biology.

Cloning, Molecular↗