Tobacco Mild Green Mosaic Virus Research Articles

A new disease in Tabernaemontana associated with Tobacco mild green mosaic virus

SummaryConspicuous viral symptoms were seen on Tabernaemontana divaricata, a member of the family Apocynaceae, grown in a commercial nursery, in Israel. The symptoms varied widely and included chlorotic ringspots and banding, oak‐leaf patterns and mosaic. At the end of the winter, large yellow spots, which later became necrotic, appeared on fully expanded leaves. The necrotic zones later fell out, leaving “shot holes”. Preliminary analysis suggested that the disease was associated with a tobamovirus. Particles typical of a tobamovirus were observed by electron microscopy only in samples taken from symptomatic leaves. Apartial segment of the 5′‐terminus of the viral RNA, which comprised of 533 bp was cloned and sequenced. Comparison of the predicted amino acid sequence with those of other tobamoviruses revealed 94% identity with Tobacco mild green mosaic virus (TMGMV) genome. Primers specific to the coat protein (CP) gene of TMGMV used in a reverse transcription‐polymerase chain reaction assay (RT‐PCR) gave an expected amplification product of 455 bp. The amino acid composition of the cloned CP gene, which shows a complete identity to that of TMGMV, confirmed the identity of TMGMV infecting Tabernaemontana. Polyclonal antibodies prepared against the virus and used in double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) enabled specific detection of the virus in crude sap extracted from T. divaricata and mechanically inoculated indicator plants. This is the first report of TMGMV infection in Tabernaemontana and the first incidence of the virus in Israel.

Heterologous Sequences Greatly Affect Foreign Gene Expression in Tobacco Mosaic Virus-Based Vectors

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence:Odontoglossumringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3′ nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3′ nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.

Specific sequence changes in the 5'-terminal region of the genome of satellite tobacco mosaic virus are required for adaptation to tobacco mosaic virus.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Problems with Interpretation of Serological Assays in a Virus Survey of Orchid Species from Puerto Rico, Ecuador, and Florida

Leaf samples collected in May 1990 from wild and cultivated orchids in Puerto Rico were tested for odontoglossum ringspot virus (ORSV), cymbidium mosaic virus (CymMV), tobacco mosaic virus common strain, tobacco mild green mosaic virus, two strains of cucumber mosaic virus, and cymbidium ringspot virus (CymRSV) with sodium dodecyl sulfate immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and/or Western blot (immunoblot) procedures. Leaf tissue from orchids cultivated in Gainesville, FL, and from the wild in Ecuador were similarly tested. No virus was detected in the 277 wild orchids, and only ORSV, CymMV, or both ORSV and CymMV were detected in 20, 73, and 22 cultivated orchids, respectively, from Puerto Rico and Florida. Several orchid plants gave ELISA reactions greater than three times the negative control with all the virus antisera tested. Other methods did not confirm the presence of virus in these plants, however. Indeed, several preimmune sera also reacted with some of these plants. Caution must be used in interpretation of low ELISA values even when these reactions are clearly greater than those of uninfected controls. These results illustrate the need to utilize more than one diagnostic technique before discarding a valuable orchid plant.

The Complete Nucleotide Sequence of Odontoglossum Ringspot Virus (Cy‐1 Strain) Genomic RNA

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy-1 strain (ORSV Cy-1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV-V(vulgare), TMV-L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5' non-coding regions of ORSV Cy-1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read-through product in which helicase-sequence and polymerase-sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV-V. The third ORF encoded a 31 K protein homologous to TMV cell-to-cell movement protein. It has 63% identities with that of TMV-V. The fourth ORF encoded an 18 K coat protein. The 5' non-coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3' non-coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.

The complete nucleotide sequence and genome organization of odontoglossum ringspot tobamovirus RNA

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot tobamovirus (ORSV) was determined. The RNA genome of ORSV is 6618 nucleotides long and contains five open reading frames (ORFs 1 to 5) coding for proteins of M(r) 126 K, 181 K, 34 K, 18 K and 52 K, respectively. This is the longest RNA of the known viruses of the Tobamovirus genus. The sequences of the ORSV RNA encoded proteins exhibit high homology to the proteins of the members of the Tobamovirus genus. The genomic organization and sequence analysis showed that ORSV is more closely related to tobacco mild green mosaic virus (TMGMV), pepper mild mottle virus (PMMV), tomato mosaic virus (ToMV) and TMV than to cucumber green mottle mosaic virus (CGMMV) and sunn-hemp mosaic virus (SHMV).

Differential Invasion by Tobamoviruses ofNicotiana megalosiphonFollowing the Hypersensitive Response

The tobamoviruses tobacco mild green mosaic virus (TMGMV) and tobacco mosaic virus (TMV) differ in their behavior in Nicotiana megalosiphon. Both viruses induce necrotic lesion formation in the inoculated leaves, but only TMV moves from the initially infected (necrotic) areas and is able to spread systemically, overcoming the hypersensitive response. The appearance and growth of necrotic local lesions induced by TMV or TMGMV in N. megalosiphon were shown to follow similar kinetics; however, virus accumulation in the local lesions was about five times greater for TMV than for TMGMV. In accordance with this, TMV replicated to much higher levels than TMGMV did in N. megalosiphon protoplasts [...]

Tobamovirus helper specificity of satellite tobacco mosaic virus involves a domain near the 5' end of the satellite genome

The molecular basis of the interactions between plant virus satellites and their helper viruses is not understood. The features of the satellite tobacco mosaic virus (STMV) genome that determine tobamovirus helper specificity were investigated using two independent strategies. The first tested the possible significance of regions of nearly identical sequence within the 3'-terminal 150 bases of the genomes of STMV and its natural helper virus, tobacco mild green mosaic virus (TMGMV). A chimeric STMV clone containing the 3' terminus of tobacco mosaic virus (TMV-U1) RNA was infectious in coinfections with TMGMV, but it did not replicate with TMV-U1. In the second strategy, populations of STMV adapted to replication with four alternative helper tobamoviruses were generated by serial passage in tobacco. RNase protection analyses of these RNA populations showed that in all cases there had been a genetic change 50 to 60 bases from the 5' terminus of the STMV genome. Similar changes were detected in several progenies of STMV clones replicated with TMV-U1, indicating that change at this site was essential for replication with a helper virus other than TMGMV. Sequence analyses of the changes at this 'helper adaptation domain' showed consistently the deletion of a single G from five consecutive Gs at bases 61 to 65. Infectivity experiments with STMV clones containing this G deletion showed that this change alone was not sufficient to modify helper specificity, so additional factors which remain to be identified must also be involved. Nevertheless, these experiments show the ability of STMV populations to undergo rapid, reproducible evolution by both selection of pre-existing variants and de novo mutation, and constitute the first molecular demonstration that the helper virus acts as a selection pressure on the evolution of satellite populations.

Genetic Structure of Natural Populations of the Plant RNA Virus Tobacco Mild Green Mosaic Virus

The statistical analysis of population subdivision in five populations of tobacco mild green mosaic virus (TMGMV) naturally infecting Nicotiana glauca has shown that the populations can be considered as samples of an almost panmictic one. In spite of the high mutation rate of this RNA virus, the genetic divergence between pairs of haplotypes, both within and between subpopulations, is very small. The observed amount of variability cannot be exclusively explained in terms of the neutral theory of molecular evolution. Under such a model, an unrealistic estimate of the effective population size equal to 86 viral particles is obtained. Positive Darwinian selection can be invoked to account for the variability observed. No direct experimental evidence of selection is presented, but, by means of both (a) high selection coefficients for driver mutations and (b) reasonable neutral and driver mutation rates for RNA viruses, the amount of variability observed can be accounted for in terms of a model of periodic, positive, Darwinian selection.

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.

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)

The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA

The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain. CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.

High genetic stability in natural populations of the plant RNA virus tobacco mild green mosaic virus

Quantitative studies on the genetic variation of plant viruses are very scarce, in spite of their theoretical and applied importance. We report here on the genetic variability of field isolates of the plant RNA virus tobacco mild green mosaic virus (TMGMV) naturally infecting the wild plantNicotiana glauca Grah. The populations studied were composed of a high number of haplotypes. Two main features are found regarding TMGMV variation: First, there is no correlation between genetic proximity of isolates and geographic proximity of the sites from which they were obtained; and second, the estimated divergence among haplotypes is low, and values are maintained regardless of the scale of the distance between the sites from which the isolates come. No comparable studies have been done with a plant RNA virus, and these two features seem to be unique for this system as compared with other RNA viruses.

Transfer of the movement protein gene between two tobamoviruses: Influence on local lesion development

The effects of transfer of the movement gene between the tobamoviruses tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) were studied. The movement protein (MP) gene of TMGMV was cloned into an infectious cDNA of TMV to build the recombinant virus V23. V23, like TMV and TMGMV, caused systemic infection in Nicotiana tabacum Xanthi. In N. sylvestris V23 and TMV spread systemically although TMGMV produces necrotic local lesions on this host. V23 and TMV cause systemic infection on tomato plants while TMGMV does not infect tomato. In Xanthi nc plants, V23 produced necrotic local lesions similar in size to those produced by TMGMV. On the other hand in transgenic Xanthi nc tobacco plants that express a gene encoding the MP of TMV the necrotic lesions produced by V23 and TMGMV were similar in size to those produced by TMV. These results indicate that the size of necrotic lesions produced by TMGMV and TMV on Xanthi nc plants is influenced by the MP gene.

A classification of the tobamoviruses based on comparisons among their 126K proteins

The products of partial proteolysis of the Mr 126,000 in vitro translation products of the RNA of eight tobamoviruses were separated by SDS-polyacrylamide gel electrophoresis. The peptide patterns obtained were compared using a computer program designed to establish phylogenetic relationships. The resulting most-parsimonious phylogenetic trees grouped the tobamoviruses into clusters I (tobacco mosaic virus, tomato mosaic virus, tobacco mild green mosaic virus, pepper mild mottle virus) and II (sunn-hemp mosaic virus, cucumber green mottle mosaic virus, kyuri green mottle mosaic virus), with ribgrass mosaic virus in an intermediate position. This clustering resembles that obtained when the coat proteins of these viruses are compared. If the tobamoviruses have arisen by divergence from an ancestral type, the results suggest that different parts of the genome have diverged similarly and that recombination has not played a major role in the evolution of the group.

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus (TMGMV) was determined. It shows 64.4% sequence homology with the genomic RNA of tobacco mosaic virus (TMV) and 66.0% with that of tomato mosaic virus (ToMV). Its genomic organization is similar to that of TMV and ToMV. The 5′ proximal open reading frame (ORF) encodes a 126K polypeptide and a 183K readthrough product in which nucleotide-binding and polymerase-sequence motifs are found. The third ORF encodes a 28.5K protein homologous to TMV and ToMV movement proteins. A conserved core is found with four other tobamoviruses and two tobraviruses suggesting a common mechanism of cell-to-cell movement for tobamo- and tobraviruses. The fourth ORF encodes the 17.5K coat protein. Homology between the RNAs of TMGMV and its satellite virus STMV is limited to their 3′ termini, and structural comparisons suggest that this region may determine the nature of the satellite/helper virus interaction.

Transgenic Tobacco Plants Expressing a Coat Protein Gene of Tobacco Mosaic Virus Are Resistant to Some Other Tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.

Bell Pepper Mottle Virus, a Distinct Tobamovirus Infecting Pepper

AbstractBell pepper mottle virus (BPeMV) can be distinguished by symptomatology and host range from other tobamoviruses but a reliable identification needs serological tests. The relationships of BPeMV to tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (PMMV) were investigated using precipitin drop tests on slides, immunodiffusion gel tests, double antibody sandwich enzyme‐linked immunosorbent assay (ELISA), and indirectELISA using enzyme‐linked goat anti‐rabbit globulins for the determination of antiserum titers and serological differentiation indices (SDI). Comparisons of SDIs and amino acid composition data demonstrated that BPeMV is a new species of the tobamovirusgroup. BPeMV, ORSV, PMMV, and TMGMV form a cluster within the genus (group) and could be considered as a subgenus of tobamoviruses.

Non‐Reciprocity of the Serological Relationship Between the Italian III Strain and Other Strains of Tobacco Mild Green Mosaic Virus

AbstractThe Italian III strain (It III) of tobacco mild green mosaic virus (TMGMV) could not be distinguished from other strains of TMGMV using three antisera to this strain in precipitin, immunodiffusion gel tests, and ELISA. Antisera to the strains U2, PTMV, G‐TAMV, and type TMGMV, however, were able to differentiate these strains from It III strain. The non‐reciprocity of the serological relationship may be explained by assuming that It III lacks a specific epitope that the other strains have or that the, corresponding region in It III is not immunogenic.