Strains Of Tobacco Mosaic Virus Research Articles

Increase of .BETA.-1,3-Glucan Hydrolase and Acquisition of Resistance in White Burley Leaves by Tobacco Mosaic Virus and Salicylate.

When the leaves of Nicotiana tabacum L. cv. White Burley were inoculated with a tomato strain of tobacco mosaic virus (TMV-T), activity of β-1, 3-glucan hydrolase was increased, but not with an ordinary strain of TMV (TMV-OM) and mechanical injury. An increase of the enzyme activity in uninoculated upper leaves of the same plants in which the lower leaves were inoculated with TMV-T, was positively correlated with acquired systemic resistance to TMV-T. Both sizes and numbers of local lesions produced on challenge-inoculated upper leaves decreased to about 30% of the control. However, when the inoculated lower leaves were cut off the 2nd day after TMV-T inoculation, no increase of the enzyme activity and systemic resistance in the upper leaves was observed. The enzyme activity was increased when detached half-leaves of the same plant were incubated on sodium salicylate solution, and showed a resistance to inoculated TMV-T. Furthermore, the enzyme activity was increased in the upper leaves of the same plant in which the lower leaves were injected with sodium salicylate and such the upper leaves showed the resistance to TMV-T. Polyacrylamide gel electrophoresis of the enzyme extracts from both the leaves inoculated with TMV-T and the leaves with induced resistance showed the presence of two isozymes of the glucan hydrolase. The involvements of salicylate on the increase of the enzyme activity and acquisition of the resistance to TMV-T are discussed.

Nucleotide Sequence of the Coat Protein and 30K Protein Genes of Tobacco Mosaic Virus-Rakkyo Strain Locally Intecting Nicotiana tabacum L. cv. Bright Yellow.

The nucleotide sequence of the rakkyo strain of tobacco mosaic virus (TMV-R) RNA corresponding to about 2, 000 nucleotides from the 3′ terminus was determined. The sequence contained the 30K protein gene, coat protein gene and 3′ non-coding region which are located in nucleotides 687 to 1493, 205 to 684 and 1 to 204 from the 3′ terminus of the genome, respectively. Sequence analysis showed that TMV-R conserved a higher degree of nucleotide sequence homology with those of TMV-common strains (-OM and -vulgare) than those of four other tobamoviruses. Comparing the deduced amino acid sequence of the coat protein between TMV-R and -OM strains, 8 substitutions were found in total 158 amino acids. The deduced 30K protein homology between TMV-R and -OM was 95.5%, showing 12 amino acid substitutions. TMV-R could efficiently infect and multiply in protoplasts of Nicotiana tabacum cv. Bright Yellow (BY), differing from the previous data using intact BY plants. The results indicated that the amino acid substitutions in the 30K protein might be associated with deficiency of function as transport protein for TMV-R in BY.

Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli.

High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4 kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2-18 h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4 micrograms/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus "self-assembly" in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.

Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance.

The genome of Ob, a tobamovirus that overcomes the N gene-mediated hypersensitive response (HR), was cloned as a cDNA, and its nucleotide sequence was determined. The genomic organization of Ob is similar to that of other tobamoviruses, consisting of 6506 nucleotides and containing at least four open reading frames. These open reading frames encode a 126-kD polypeptide with a 183-kD readthrough product, a 30.6-kD movement protein, and an 18-kD coat protein. A bacteriophage T7 promoter sequence was fused to the full-length cDNA clone to obtain infectious RNA transcripts. These transcripts, when inoculated onto tobacco plants, induced disease symptoms indistinguishable from plants inoculated with Ob viral RNA. To determine which viral factor is responsible for the resistance-breaking character of Ob, a recombinant virus was constructed in which the movement protein gene of tobacco mosaic virus was replaced with that of Ob. Cultivar Xanthi NN tobacco plants infected with this virus responded with an HR, indicating that the Ob movement protein alone does not act to overcome the N gene-mediated response. Following mutagenesis of the infectious Ob cDNA clone with hydroxylamine, populations of transcripts from the mutagenized DNA were inoculated onto Xanthi NN tobacco, and a variant that induced the HR was identified. The mutant was analyzed and found to contain a single nucleotide change in the 126-kD gene. Recreating the mutation in the Ob cDNA clone by site-directed mutagenesis resulted in a virus that caused symptoms identical to the chemically induced mutant.

Analysis of a Tobacco Mosaic Virus Strain Capable of Overcoming N Gene-Mediated Resistance

Analysis of a Tobacco Mosaic Virus Strain Capable of Overcoming N Gene-Mediated Resistance

On the relationship between X-bodies and symptom development in plants infected with different tobamoviruses.

The relationship between systemic mosaic symptoms and the occurrence of viral 126-kDa protein in X-bodies was studied in tobacco infected with the tobacco mild green mosaic virus (TMGMV) strains U2, U5, and ribgrass mosaic virus (RMV) strain HR, and in other plant species infected with tobacco mosaic virus (TMV) strain W U 1. Strains U2, U5, and HR coded for proteins of 126, 126, and 130 kDa, respectively, but these were not recognized by antisera against the corresponding protein from W U 1. Only the HR 130-kDa protein reacted with an antiserum raised against a peptide of amino acids 849-863 from the sequence of W U 1. Electron microscopic analysis established the presence of virus clusters in the cytoplasm, as well as in chloroplasts, in leaf tissue infected with U 2 or U 5, and adjacent to nuclei and chloroplasts in scattered cells infected with HR. X-bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U 1-infected tomato displaying severe mosaic symptoms. Large X-bodies were detected near nuclei in W U 1-infected tomato displaying severe mosaic symptoms, but none were detected after infection of tobacco with any of the other tobamoviruses. The induction of X-bodies appears to be characteristic of some tobamovirus only and, at best, can only be associated with, rather than causative of, the severity of symptoms induced by those viruses.

Preparative immunoabsorption electrophoresis. 1978.

A method for separating a component from a mixture of antigens is described. The component, which may be a virus or subfraction of a virus, is isolated by driving the mixture by electrophoresis through a gel containing precipitating antibodies directed against the unwanted components. The method is illustrated by the isolation of hepatitis B antigen from whole serum and by the separation of wild cucumber mosaic virus from a strain of tobacco mosaic virus.

Activity of .BETA.-1,3-Glucan Hydrolase and Acquired Resistance in the Upper Leaves of Nicotiana glutinosa Followed by Infection with Tobacco Mosaic Virus.

When the lower leaves of Nicotiana glutinosa were inoculated with an ordinary strain of tobacco mosaic virus (TMV), β-1, 3-glucan hydrolase activity increased in the upper leaves of the same plant. The increase in enzyme activity was first detected 4 days after inoculation of the lower leaves, reaching a maximum of 6 times higher than the mock-inoculated control plants 10 days after the inoculation. The enzyme activities of the intercellular fluid (ICF) and of the rest of the tissue homogenates of both the inoculated lower leaves and the uninfected upper leaves of the plant increased with the progress of infection. The enzyme activity of ICF in the inoculated lower leaves was 6-10% of the total activity and in the upper leaves 7-11%. Acquired systemic resistance in the upper leaves to TMV appeared 4 days after inoculation of the lower leaves with TMV. Ten days after inoculation the size of local lesions in the upper, virus-challenged leaves decreased to 50% of the control. However, when the inoculated lower leaves were cut off the 2nd day after inoculation, no increase of the enzyme activity in the upper leaves was detected and no acquired resistance to TMV was observed. The relationship between β-1, 3-glucan hydrolase activity and the acquired resistance to TMV-infection is discussed.

Impeded Phloem-Dependent Accumulation of the Masked Strain of Tobacco Mosaic Virus

Impeded Phloem-Dependent Accumulation of the Masked Strain of Tobacco Mosaic Virus

Capsid protein gene sequences of four tobacco mosaic virus strains defective for virus assembly.

Capsid protein gene sequences of four tobacco mosaic virus strains defective for virus assembly.

Accumulation of viral 126 kDa protein and symptom expression in tobacco systemically infected with different strains of tobacco mosaic virus

Tobacco mosaic virus strains W U1, flavum, V4 and masked, which induce symptoms in tobacco ranging from a severe mosaic to virtually no reaction, were used to study relationships between accumulation of viral 126 kDa protein, X-body formation and symptom expression. The RNAs of all strains encode proteins of similar sizes to those of the common strain W U1. Antisera against the coat and 126 kDa proteins of W U1 recognized corresponding proteins of the other three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coat protein in virus crystals. The 126 kDa protein was localized in large X-bodies associated with nuclei in W U1- and flavum-infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked-infected plants. Neither X-bodies, nor 126 kDa proteins were observed in V4-infected tissue. Since in vitro synthesized 126 kDa protein from V4 was not more sensitive to proteolysis than that from W U1 or flavum, accumulation of the protein in X-bodies in the latter seems to result from excess synthesis. The possibility that the X-bodies are involved in the expression of severe symptoms is discussed.

Structure of the U2 strain of tobacco mosaic virus refined at 3.5 Å resolution using X-ray fiber diffraction

The structure of the U2 strain of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 3.5 Å resolution, and refined by a combination of restrained leastsquares and molecular dynamics methods to an R-factor of 0.096. The structure is extremely similar to that of the common strain of TMV, with the largest differences being in the protein loop that makes up the inner surface of the virus, and in the C-terminal region on the outer surface. Differences in the inner loop can be correlated with differences in the properties of the two viruses.

Host Effect on Cross Protection Between Two Strains of Tobacco Mosaic Virus

AbstractThe expression of cross protection between two strains of tobacco mosaic virus (TMV‐C and TMV‐P) differed in Arabidopsis thaliana cv. Columbia and Nicotiana tabacum cvs Samsun and Xanthi. Protection in A. thaliana cv. Columbia was expressed as a prevention of systemic movement of the challenge strain, regardless of the protecting strain of TMV, Protection in N. tabacum cvs Samsun and Xanthi was expressed as an inhibition of an early event in the infection process. The results presented indicate that the host may influence the mechanism by which cross protection is expressed between the same virus strains.

Analysis of genetic heterogeneity within the type strain of satellite tobacco mosaic virus reveals several variants and a strong bias for G to A substitution mutations

Satellite tobacco mosaic virus (STMV) is a small plant virus that is dependent for its replication on the presence of a helper tobamovirus. RNase protection analysis of genomic RNA of the STMV type strain revealed that it was composed of two major genome types which differed at a single detectable site near nucleotide 753. Analyses of 42 full-length STMV clones for sequence heterogeneity resulted in the identification of 16 variants distinguishable by unique RNase protection assay patterns. Characterization of these variants confirmed the presence of a major heterogeneity site at nucleotide 751 and identified several sites of sequence micro heterogeneity typical of an RNA quasispecies population. Mapping of the heterogeneity sites revealed an apparently random distribution along the length of the STMV genome, with no significant clustering or preference for noncoding regions. Infectivity experiments in tobacco showed that RNA transcripts of 13 of the 16 variant clones were infectious, indicating that most of the variants represent functional genomes coexisting in the type strain with the two major genome types. Sequence analyses revealed that most of the heterogeneity sites detected, including the major site of heterogeneity, were single base differences. Assessment of all the heterogeneity sites found in the total of 10,545 nt sequenced allowed us to estimate that the RNase protection assays detected approximately 50% of the differences present in the 16 clones studied. The nature of these differences was highly biased in that 18 of the 29 single base differences characterized (62%) were G to A substitutions.

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA.

Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.

Fluctuations of Particles on Chloroplast Thylakoid Membranes in Tomato Plants Infected with Virulent or Attenuated Strain of Tobacco Mosaic Virus.

Fluctuations of Particles on Chloroplast Thylakoid Membranes in Tomato Plants Infected with Virulent or Attenuated Strain of Tobacco Mosaic Virus.

Field Resistance of Transgenic Tomatoes Expressing the Tobacco Mosaic Virus or Tomato Mosaic Virus Coat Protein Genes

Under field conditions, transgenic tomato plants that express the coat protein (CP) gene of common (U1) strain of tobacco mosaic virus (TMV) showed a high degree of resistance to the U1 strain and to a more severe strain of TMV, PV230. Tomato fruit yields of inoculated control plants decreased 20% with U1 and 69% with PV230, whereas the CP+ line did not show any yield reduction after inoculation with U1 or PV230. In contrast, tomato plants expressing TMV CP showed either a low level of resistance or no resistance to several different tomato mosaic virus (ToMV) strains in the field (...)

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.

Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.

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.