Strains Of Tobacco Mosaic Virus Research Articles

Breakdown of Cross Protection Between Strains of Tobacco Mosaic Virus Due to Susceptibility of Dark Green Areas to Superinfection

Reciprocal cross protection between the common strain of tobacco mosaic virus (TMV-C) and TMV-P (a necrotizing strain in Nicotiana sylvestris) was investigated in plants of N. tabacum Samsun' and Xanthi'. When a concentration of challenge inoculum of 1 μg/ml or higher of either TMV-P or TMV-C was used, there was superinfection of plants infected with the heterologous strain. The susceptibility to superinfection was associated with dark green areas of mosaic leaves, which were more susceptible to superinfection than the neighboring light green areas (...)

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°) 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-non permissive temperature (33°) 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°. 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.

Introduction of Tobacco Mosaic Virus-OM RNA into Free Cells of Brassica campestris L. cv. Komatsuna by Electroporation.

The electroporation method was applied to introduce ordinary strain of tobacco mosaic virus (TMV-OM) RNA into free cells of Brassica campestris L. cv. Komatsuna, a natural non-host plant of TMV. The infection rate of the free cells was as low as 30% when a mixture of free cells and TMV-OM RNA was electroporated. However, the rate was enhanced to about 70% when the mixture was preincubated in an ice bath for 60min prior to electroporation, demonstrating that this virus could multiply in Komatsuna cells. On the other hand, fluorescent microscopy of thin sections of Komatsuna leaves inoculated with TMV-OM revealed that this virus could multiply in inoculated leaves without any visible symptoms but did not transfer to upper leaves. Considering the fact that cauliflower mosaic virus (CaMV) can infect with a higher rate in its host plant, Komatsuna, giving a typical symptom, it is likely that the degree of transfer and/or multiplication of TMV-OM in Komatsuna tissues might be much lower than that of CaMV in some reason, or that symptom appearance might be regulated by a different mechanism from that of viral multiplication.

Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus

Tobacco (Nicotiana tabacum L. cv. Samsun) plants inoculated with different strains of tobacco mosaic virus (TMV) inducing mosaic symptoms of widely varying severity were studied with in vivo chlorophyll fluorescence. This method was used to deduce photosynthetic electron transport efficiency in relation to symptom expression. The quantum yields of photosystem II (PS II) electron transport rate were significantly diminished in virus strains inducing loss of chlorophyll. The reduction in young mosaic‐diseased leaves appeared to be due in part to a reduction in the fraction of open reaction centers, whereas in older leaves exhibiting less pronounced symptoms the reduction was mainly caused by a reduced efficiency of capture of excitation energy of open PS II reaction centers. Upon infection with any of the five virus strains PS II seemed to be irreversibly damaged in the inoculated leaves and the ones directly above, indicative of a possible increased susceptibility to photoinhibition in these leaves (Somersalo and Krause 1989) even when no symptoms were apparent. Symptom expression did not appear to be related to the influence of the virus on PS II activity, because the severest effects occurred in the inoculated leaves, which either remained symptomless or developed slight yellowing only. This study demonstrates the usefulness or modulated chlorophyll fluorescence measurements for the investigation of plant‐virus interactions. It is particularly important when visual symptoms are absent.

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.

Characterization of the Masked Strain of Tobacco Mosaic Virus: Identification of the Region Responsible for Symptom Attenuation by Analysis of an Infectious cDNA Clone

A strain of tobacco mosaic virus (TMV) that produces mild (attenuated) symptoms on tobacco plants has been molecularly cloned to identify the region of the genome responsible for symptom attenuation. A full-length cDNA clone whose transcripts produce the parental disease phenotype on both systemic and hypersensitive host plants has been constructed. This infectious clone was sequenced, and 55 base changes relative to the published sequence of common TMV (strain U1) were identified. These changes resulted in 12 amino acid alterations in the open reading frames encoding the 126/183-kDa and 30-kDa movement proteins; two of these changes were determined not to be responsible for the attenuated phenotype. Exchange of restriction fragments between the infectious mild strain cDNA and an infectious U1 strain cDNA indicated that the determinants involved in symptom attenuation reside in the open reading frame encoding the 126/183-kDa proteins of TMV; these proteins are involved in viral replication.

Practical detection of wasabi strain of tobacco mosaic virus by using "cocktail" monoclonal antibodies.

Nine monoclonal antibodies (MABs) were examined for their reactivities to wasabi strain of tobacco mosaic virus (TMV-W) in two indirect ELISA and competitive ELISA. Based on the results, seven epitopes were discriminated on the surface of a particle of TMV-W and one cryptotope was found inside of the virus particle. For practical detection of TMV-W, mixture of different types of MABs, i.e., cocktail MABs, were examined in double sandwich antibody form ELISA (DAS-ELISA). Five MABs with high titers, which recognized different epitopes were chosen to compound the cocktail MABs. Among the combinations of five MABs, that with the highest sensitivity was selected for coating γ-globulin. To obtain the sensitive and specific conjugates, four MABs were used for compounding cocktail conjugate. When the MABs which did not compete in the reaction with antigens were compounded, the highest sensitivity was obtained. When the cocktail γ-globulin and cocktail conjugates were used for detection of TMV-W from infected plants in the DAS-ELISA, the sensitivity was almost the same as that used polyclonal antibodies. Furthermore, the cocktail MABs distinguished TMV-W from crucifer strain of TMV, the differentiation of which was difficult by using polyclonal antibodies.

Serological properties of wasabi strain of tobacco mosaic virus.

Serological properties of wasabi strain of tobacco mosaic virus.

Comparison of virus production in infected plants between an attenuated tomato strain (L11A) and its wild strain (L) of tobacco mosaic virus.

Comparison of virus production in infected plants between an attenuated tomato strain (L11A) and its wild strain (L) of tobacco mosaic virus.

Regulation of Tobamovirus Gene Expression

Publisher SummaryThis chapter discusses tobacco mosaic virus (TMV) strains U1, OM, L, CGMMV, 0, and Cc. The production of each TMV protein is regulated differently, both in amounts and times of production. The chapter discusses some of the strategies that tobamoviruses uses to control gene expression: (1) different subgenomic RNA promoter/leader sequences control timing of expression of genes, (2) genes expressed via subgenomic mRNAs are expressed in decreasing amounts with increasing distances from the 3' terminus, and (3) TMV mRNAs appear to be translationally regulated differently from host mRNAs. Genome organization affects gene expression, but it appears to be equally important for the efficiency of replication and the ability of the genomic structure to be stably propagated. Different virus groups have evolved different gene arrangements. Tobamovirus genes expressed via subgenomic mRNAs appear to be expressed in increasing amounts when positioned nearer the 3’ terminus.

Infection of suspension-cultured cells of carrot with tobacco mosaic virus

The type strain of tobacco mosaic virus (TMV) was found to infect suspension-cultured cells of wild carrot ( Daucus carota L.) in the absence of mechanical inoculation procedures. The course of infection was followed by enzyme-linked immunosorbent assay (ELISA) over a 21-day cellular growth period. The kinetics of TMV antigen accumulation by the cells were dependent on the growth cycle stage of the latter at the time of transfer to fresh medium containing TMV and on the hormonal constitution of the growth medium. The nature of the growth medium also influenced the morphology and state of differentiation of the cells, although such effects could not be related directly to antigen accumulation. Under certain conditions two phases of antigen accumulation were observed separated by a decline in antigen level, and thus the cells may develop transient anti-viral activity.

Genetic heterogeneity of the RNA genome population of the plant virus U5-TMV

The genetic heterogeneity in a population of the U5 strain of tobacco mosaic virus (U5-TMV) was studied. The T1 fingerprint characterizing a cloned population did not vary after a new cloning step in the local lesion host Nicotiana tabacum Xanthinc, nor during four series of 20 passages in the systemic host N. tabacum Samsum. No heterogeneity was observed among 10 clones derived from the cloned populations, while 1 of 18 clones derived from a 20-fold passaged population differed from the rest in 1 of 55 oligonucleotides. A higher heterogeneity was found in an uncloned field isolate in which 2 of 10 clones differed in type in 1 and 2 oligonucleotides, respectively. These data agree with those reported for bacterial and animal RNA viruses and are compatible with the quasi-species model for RNA populations. On the other hand, the intrapopulational heterogeneities found for U5-TMV are considerably smaller than those reported for other RNA viruses, our data showing a high genetic stability for U5-TMV.

Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato.

A resistance-breaking strain of tobacco mosaic virus (TMV), Ltb1, is able to multiply in tomatoes with the Tm-2 gene, unlike its parent strain, L. Nucleotide sequence analysis of Ltb1 RNA revealed two amino acid changes in the 30-kD protein: from Cys68 to Phe and from Glu133 to Lys (from L to Ltb1). Strains with these two changes generated in vitro multiplied in tomatoes with the Tm-2 gene and induced essentially the same symptoms as those caused by Ltb1. Strains with either one of the two changes did not overcome the resistance as efficiently as Ltb1, although increased levels of multiplication were observed compared with the L strain. Results showed that both mutations are involved in the resistance-breaking property of Ltb1. Sequence analysis indicated that another resistance-breaking strain and its parent strain had two amino acid changes in the 30-kD protein: from Glu52 to Lys and from Glu133 to Lys. The fact that the amino acid changes occurred in or near the well conserved regions in the 30-kD protein suggests that the mechanism of Tm-2 resistance may be closely related to the fundamental function of the 30-kD protein, presumably in cell-to-cell movement.

Accumulation of necrotic lesion inducing variants in TMV-infected plantlets derived from leaf disks of Nicotiana sylvestris

In an isolate of tobacco mosaic virus strain U1 there exists a small subpopulation containing a variant strain of the virus that induces the hypersensitive response on Nicotiana sylvestris Spegazzini & Comes. This type of variant is strongly selected for during the regeneration of plantlets from mature leaf tissue of plants infected with tobacco mosaic virus U1. When whole plants derived from disks were transferred into a glasshouse, those containing this type of variant were severely stunted, showed mosaic symptoms, and most of them died. Some that had originally contained lower titers of variant-type virus survived to flower but produced only a few seeds. Plants that initially contained only wild-type virus had high titers of tobacco mosaic virus, survived and grew well, exhibited mosaic symtoms, and flowered and set seed normally. Repeated assays of virus in these plants revealed no detectable variant-type virus. Apparently, during callus development and organogenesis in culture, partial segregation of the mixed U1 population occurred, and variants preferentially infected the developing tissues. This represents a situation in which dramatic change in the genetic structure of an RNA virus population occurs during development of the host plant.

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: An effect of the viral coat protein

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced F v/ F m ratio for chlorophyll fluorescence kinetics, at 25°C. The photosystem II (PS Il)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.

Characteristics of epitopes on the tomato strain of tobacco mosaic virus detected by monoclonal antibodies.

Eleven murine monoclonal antibodies (MABs) specific for L11A, a tomato strain of tobacco mosaic virus (TMV-L11A), were produced in culture supernatants and in ascitic fluids. Reactivities of all the 11MABs with tobamovirus strains were investigated by two indirect ELISA procedures, competitive ELISA, precipitin ring interface test and immuno-double-diffusion test. Five MABs among them reacted only with tomato strains (L11A, L and CH2) and the others reacted not only with tomato strains but also with other strains [TMV-OM, P, W and cucumber strain of cucumber green mottle mosaic virus (CGMMV-Cu)]. Based on these results, at least eight epitopes were distinguished on the surface of the particles of L11A. The epitopes, except for one detected by one of the MABs (6F11), were all neotopes, and 10 out of 11MABs were anti-neotope antibodies including a heterospecific antibody (8C8). A superior epitope which reacted mainly in the antiserum was identified within these epitopes. The high specificity of the MABs showed that they were useful for the differentiation of tobamovirus strains. The titers of three MABs in ascitic fluids were 1:2, 560 and they could be substituted for rabbit antisera. This study showed that the antigenic specificity of the strains was mainly dependent on the neotopes.

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.

Ultrastructural observations on chloroplasts in tomato plants infected with an attenuated strain (L11A) of tobacco mosaic virus.

Leaves of tomato plants (Lycopersicon esculentum cv. Fukuju no.2) inoculated with TMV-L11A (attenuated) or TMV-L (virulent) were compared by electron microscopy during the course of virus infection. Estimates were also made of the numbers of virions, the chlorophyll (a+b) content and the chlorophyll (a/b) ratio. Five days after inoculation, no differences were found in chloroplast structure, chlorophyll content or chlorophyll (a/b) ratio between healthy and infected plants, although a large number of virions were observed in these plants with both strains. From 10 to 30 days post-inoculation, changes in chlorophyll content and chlorophyll (a/b) ratios became apparent, the amount being in the order healthy>L11A>L. Virion accumulation was also much greater in cells infected with L than that with L11A. In both cases, chloroplasts were swollen and finally often ruptured. Their lamellar systems, however, remained essentially undamaged. Even though the L11A never induced mosaic symptoms, this attenuated strain also caused some ultrastructural changes in chloroplasts.

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.