Tobacco Necrosis Virus Strain Research Articles

Priming crop plants with rosemary (Salvia rosmarinus Spenn, syn Rosmarinus officinalis L.) extract triggers protective defense response against pathogens

Plant bioactive compounds provide novel straightforward approaches to control plant diseases. Rosemary (Salvia rosmarinus)-derived extracts carry many prominent pharmacological activities, including antimicrobial and antioxidant, mainly due to its phenolic compounds, rosmarinic acid (RA), carnosic acid and carnosol. However, the effects of these extracts on plant diseases are still unknown, which constrains its potential application as bioprotectant in the agricultural production. In this study we demonstrate the antiviral effect of the aqueous rosemary extract (ARE) against tobacco necrosis virus strain A (TNVA) in ARE-treated tobacco (Nicotiana tabacum) plants. Our results show that ARE-treatment enhances plant defense response, contributing to reduce virus replication and systemic movement in tobacco plants. RA, the main phenolic compound detected in this extract, is one of the main inducers of TNVA control. The ARE-induced protection in TNVA-infected plants was characterized by the expression of H2O2 scavengers and defense-related genes, involving salicylic acid- and jasmonic acid-regulated pathways. Furthermore, treatment with ARE in lemon (Citrus limon) and soybean (Glycine max) leaves protects the plants against Xanthomonas citri subsp. citri and Diaporthe phaseolorum var. meridionalis, respectively. Additionally, ARE treatment also promotes growth and development, suggesting a biostimulant activity in soybean. These results open the way for the potential use of ARE as a bioprotective agent in disease management.

Investigation of Novel RNA Elements in the 3'UTR of Tobacco Necrosis Virus-D.

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3′ untranslated region (3′UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3′UTR, the sequence located upstream of the 3′-cap independent translation enhancer (3′CITE). Our results indicate that (i) a long-range RNA–RNA interaction between an internal RNA element and the 3′UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3′CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3′UTR of the TNV-D genome that are important for virus survival.

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus (STMV) and the strains of satellite tobacco necrosis virus (STNV), STNV-C, STNV-1, STNV-2, Escherichia phages MS2, ϕX174, α3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs.

Isolation and characterization of <i>tobacco necrosis virus</i> detected on some vegetable species

* Corresponding author. E-mail: irena.zitikaite@botanika.lt A virus disease agent was isolated from greenhouse-grown cucumber (Cucumis sativus L.), field-grown common bean (Phaseolus vulgaris L.) and white cabbage (Brassica oleracea var. capitata (L.) Alef) crops in Lithuania. The diseased vegetable plants expressed various spotting or mottling symptoms on leaves or fruits and necrotic symptoms on older leaves. The objective of this study was to identify the causal agent of the virus-like disease of these plant species. For the identification of the causal virus, the infection from cucumber, common bean and white cabbage leaves or fruits was transmitted to main test plant species which reacted by only local lesions. In electron microscopic preparations from infected plants, only icosahedral virus particles about 26 nm in diameter were detected. According to particle morphology, host range and symptoms, the causal virus was identified as tobacco necrosis virus (TNV). This finding was supported by the reverse transcription–polymerase chain reaction (RT–PCR) technique which confirmed that the virus isolates from the Lithuanian cucumber, common bean and white cabbage crops were identical with the TNV strain A “Nebraska” isolate.

Complete nucleotide sequence of a new strain of Tobacco necrosis virus A infecting soybean in China and infectivity of its full-length cDNA clone

The complete nucleotide sequence of a virus isolated from soybean (Glycine max (L.) Merr.) in China, previously identified as a new strain of Tobacco necrosis virus A (TNV-A) based on its biological, serological properties, and coat protein (CP) sequence and named as TNV-A C, was determined and compared with that of TNV-A and other closely related Necroviruses and Carmoviruses. The viral RNA genome consists of 3,682 nucleotides and contains five open reading frames (ORFs). TNV-A C showed 86.4% overall nucleotide sequence identity to TNV-A. The CP and putative RNA-dependent RNA polymerase (RdRp) showed 88.8 and 95.9% amino acid identity, respectively, to that of TNV-A. The greatest difference between TNV-A C and TNV-A was in the 3' terminal region: the p7K ORF region present in TNV-A was absent in TNV-A C. Phylogenetic analysis of RdRp, CP, and small ORF regions of Necroviruses confirmed TNV-A C as a new strain of TNV-A. A full-length cDNA clone of TNV-A C was constructed and used as template for run-off transcription based on the obtained sequence. The results indicate that the in vitro-synthesized viral RNA faithfully represented the biological activity of wild-type TNV-A C.

The 3' untranslated region of tobacco necrosis virus RNA contains a barley yellow dwarf virus-like cap-independent translation element.

RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.

Isolation of an RNA‐dependent RNA Polymerase fromNicotiana benthamianaPlants Infected withTobacco Necrosis Virus(strain D) and Template‐dependent Transcription of its Satellite,Satellite Tobacco Necrosis Virus(strain C) RNA

Partially purified RNA‐dependent RNA polymerase activity isolated fromNicotiana benthamianaplants infected withTobacco necrosis virusstrain D (TNV‐D) is capable of template‐dependent synthesis ofSatellite tobacco necrosis virusstrain C (STNV‐C) RNA. Thisin vitrosystem will be of great value in investigations on both TNV‐D and STNV‐C RNA replication.

Primary structure and phylogenetic analysis of the coat protein of a Toyama isolate of tobacco necrosis virus.

The amino acid sequence of the coat protein (CP) of a tobacco necrosis virus (TNV) strain, Toyama isolate, was determined by a combination of peptide and cDNA sequencing. The deduced sequence of 276 residues was compared with CPs of other TNV isolates and other plant virus isolates of Tombusviridae. It showed the highest similarity to the TNV Nebraska isolate with 92% identity and moderate similarity to the TNV strain A with 51% identity, confirming the previous serological analysis. It also showed overall similarity with CPs of mostly genera Necrovirus and Sobemovirus, and partial similarity with CPs of genera Tombusvirus and Carmovirus. Among 13 CPs that showed overall similarity, there were 10 completely conserved residues. These included three residues that participate in Ca2+ ligation at the interfaces of virion subunits in TNV crystal structure, suggesting that similar metal binding occur in the viruses of genera Necrovirus and Sobemovirus.

Mutation Analysis of cis-Elements in the 3′- and 5′-Untranslated Regions of Satellite Tobacco Necrosis Virus Strain C RNA

The putative, 3′-terminal stem-loop structure in satellite tobacco necrosis virus strain C (STNV-C) RNA constitutes an essential cis-acting structure for the promotion of negative-strand RNA synthesis and a single-stranded tail is also important. The putative, 5′-terminal stem-loop structure in STNV-C RNA is not essential for productive, plus-strand RNA accumulation but is required for optimal accumulation. Residues 2 and 3 are the minimal cis-acting sequences required for RNA synthesis. The RNA of chimeric mutants, which exchanged 3′- and 5′-untranslated regions between STNV-C and helper tobacco necrosis virus strain D RNAs, accumulated in protoplasts, implying similar replication mechanisms for both RNAs.

The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.

The complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Location of the 5′ Termini of Tobacco Necrosis Virus Strain D Subgenomic mRNAs

AbstractNorthern blot analysis of double‐stranded (ds) RNA from bean‐leaf tissue infected with tobacco necrosis virus strain D (TNV‐D) detected the 4 kb genomic RNA and two subgenomic RNAs of about 1.5 kb and 1.2 kb; RNA extracted from virus particles only contained the genomic species. Blotting and probing with a range of probes indicated the approximate locations of the 5’ends of subgenomic RNA so that primers to fine‐map the ends could be designed. When both singlestranded and ds RNA, extracted from TNV‐D infected and healthy bean leaves were used as templates for primer extension using primers complementary to sequences at, or upstream of, the initiation codons of, respectively, the coat protein and the p7a genes, major infectionspecific products were detected. Both subgenomic RNAs start at G residues. The larger subgenomic RNA is 1547 nucleotides in length with a leader sequences of 36 nucleotides upstream of the p7a gene, and the smaller subgenomic RNA has a 90 nucleotide leader upstream of the coat protein AUG and is 1202 nucteotides long. An analysis of the 5’terminal locations of both subgenomic RNAs and the previously mapped analogous subgenomic RNAs associated with infection with the related TNV‐A isolate, revealed a marked degree of homology downstream of the initiation sites for each RNA. This homology was maintained at the 5’termini of both virion RNAs and could be extended to another isolate of TNV for which partial sequence data, but not subgenomic mapping RNA data are available.

Subgenomic RNAs mediate expression of cistrons located internally on the genomic RNA of tobacco necrosis virus strain A.

Upon infection of tobacco protoplasts, the genomic RNA of tobacco necrosis virus strain A (TNV-A) accumulates linearly in time. The accumulation patterns of the two subgenomic RNAs resemble those of endogenous mRNAs in that the peak levels are reached after several hours. The accumulation of the 1.3-kb subgenomic RNA is delayed by 1 h compared with that of the 1.6-kb subgenomic RNA, which illustrates the important role of the subgenomic RNAs in the regulation of TNV-A gene expression. The locations of the 5' nucleotides of the subgenomic RNAs reveal that the 5'-proximal cistrons of the 1.6- and 1.3-kb RNAs encode an 8-kDa protein from open reading frame (ORF) 3 and the coat protein from ORF 5, respectively. In a wheat germ translation system, a synthetic transcript resembling the 1.6-kb RNA expresses both ORFs 3 and 4. Moreover, the synthesis of the 6-kDa protein from ORF 4 depends on the translation efficiency of ORF 3, suggesting that in vivo, ORFs 3 and 4 are both expressed from the 1.6-kb RNA. The major in vitro translation product of TNV-A genomic RNA is the coat protein. We show that the region upstream of the coat protein promotes internal initiation of translation in vitro. However, this region is functionally inactive in vivo, suggesting that TNV-A genomic RNA is not important for coat protein synthesis in plants.

The complete nucleotide sequence of tobacco necrosis virus strain D

The complete sequence of the RNA genome of tobacco necrosis virus strain D (TNV-D) consisting of 3759 nucleotides has been determined. The positive strand contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 22K protein terminating with an amber codon which may be read through to produce a 82K protein (p82). Two small centrally located ORFs each encode two out-of-frame 7K proteins (p7a and p7b). The 3'-proximal ORF encodes the 29K coat protein (CP), the N terminus of which has been sequenced directly. The genomic organization of TNV-D is very similar to that of TNV-A but differs in the placement of the p7a ORF, which does not overlap the p82 ORF in TNV-D, and in the absence of an ORF downstream of the CP gene in TNV-D. The p82 ORF shows extensive sequence similarity with the putative polymerases of the carmovirus group. This ORF is also as closely related to the corresponding ORF of TNV-A as it is to the corresponding ORF of the tombusvirus cucumber necrosis virus. The amino acid sequence of the TNV-D CP gene is similar to both the TNV-A and southern bean mosaic virus CP genes. Of the two p7 ORFs, p7a exhibits amino acid sequence similarity with corresponding proteins from TNV-A, melon necrotic spot virus, carnation mottle virus, turnip crinkle virus and maize chlorotic mottle virus, whereas the p7b ORF appears to be unique to TNV-A and TNV-D. Only the 3'-terminal three nucleotides of TNV-D genomic RNA are identical to the 3'-terminal nucleotides of the TNV satellite virus.

Genome structure of tobacco necrosis virus strain A

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5′-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5′ end no homology was found with the RNA of STNV (satellite of TNV).

Characterisation of a new strain of tobacco necrosis virus isolated from nutrient feeding solution*

SummaryA virus, isolated from the nutrient feeding solution of a bell pepper culture in a glasshouse, was characterised as a new serotype of tobacco necrosis necrovirus (TNV) and named TNV‐nft. The virus was monopartite and contained a single RNA species and a single coat protein with molecular weights of 1.45± 106 and 2.9 ± 104 daltons, respectively. The particle had an apparent sedimentation coefficient of 110 S and a density of 1.37 g/cm3 in cesium chloride. In decoration tests antiserum prepared against TNV‐nft reacted most strongly with the TNV strains Kassanis A and B, but at lower dilutions it also reacted with all other TNV‐serotypes available. In Ouchterlony tests spur formation occurred with TNV strains Kassanis A and B, indicating that the TNV‐nft strain was serologically distinct. The isolate was not associated with a satellite virus. Cloned cDNA prepared against viral RNA reacted exclusively with the viral RNA band in Northern blots.

Detection of Petunia Asteroid Mosaic, Carnation Ringspot and Tobacco Necrosis Viruses in Ditches and Drainage Canals in a Grapevine‐Growing Area in West Germany

AbstractA number of virus isolates were obtained from ditches and drainage canals in the Palatinate grapevine‐growing area (‘Rheinpfalz’) in West Germany. By means of the immunoelectron microscopical decoration and the agar gel double diffusion tests these isolates were identified as petunia asteroid mosaic virus, carnation ringspot virus and tobacco necrosis virus strain D, respectively.

Chenopodium necrosis: a distinctive strain of tobacco necrosis virus isolated from river water

SUMMARYA distinctive strain of tobacco necrosis virus (TNV) of unknown source was repeatedly isolated from water of the River Avon (Warwickshire) and two of its tributaries (R. Swift and R. Alne) using a technique developed for the concentration and isolation of water‐borne bacteriophages. The same strain was isolated from the rivers Cam and Thames and from Lake Esthwaite (Cumbria) together with tomato bushy stunt virus.The TNV strain, designated Chenopodium necrosis (TNV‐CN) was mechanically transmissible to C. amaranticolor and C. quinoa in both of which it caused local lesions and systemic infection. TNV‐CN caused no infection when inoculated to tobacco (Nicotiana tabacum cv. White Burley) plants.The virus was not adsorbed to soil, could be isolated from leachate of soil in which systemically infected C. quinoa were grown and C. quinoa plants became infected when grown in soil watered with suspensions of the virus.The virus was not transmitted by Myzus persicae but was vectored by the zoospores of a lettuce isolate of Olpidium brassicae.TNV‐CN was infective after 10 min at 85 °C., 3 wk at 20 °C and when diluted to 10‐8 but not 10‐9. Purified virus preparations contained c. 26 nm isometric virus particles.TNV‐CN contained single‐stranded RNA (mol. wt 1·5 × 106) and one protein (mol. wt c. 26·4 × 103) which co‐electrophoresed in polyacrylamide gels with the protein of the D strain of TNV (TNV‐D). Analytical centrifugation of TNV‐CN indicated a single component virus with the same sedimentation coefficient (s20, w= 115S) and buoyant density (1·385) in a CsCl gradient as those of TNV‐D. TNV‐CN and TNV‐D were indistinguishable serologically.

Some Effects of pH, Salt, Urea, Ethanediol and Sodium Dodecyl Sulphate on Tobacco Necrosis Virus

SUMMARY The sedimentation coefficients of tobacco necrosis virus strain D (TNV d ) in sodium phosphate at pH 5 and pH 7 are 104S and 97S respectively. The change in sedimentation coefficient between pH 5 and 7 is fully reversible and the decrease on transfer to pH 7 is unaffected by the presence of Mg2+. TNV d is disrupted by Bacillus subtilis protease at pH 7.5 but not at pH 5; ribonuclease neither disrupts virions nor destroys the infectivity of TNV d at either pH 7.5 or pH 5. Virions are markedly unstable at alkaline pH but the effectiveness with which different buffers dissociate TNV d varies. Virus is largely resistant to dissociation by 1 m-NaCl, 1 m-urea, 10 m-ethanediol and 1% sodium dodecyl sulphate (SDS) at or below pH 6 and is largely susceptible to dissociation by these agents at or above pH 7. Our results suggest that as the pH is lowered some non-covalent bonds important for virion stability may be formed and/or some electronic repellent interactions causing virion instability may be weakened.

Some Effects of Temperature on the Early Stages of Tobacco Necrosis Virus Multiplication

SUMMARY The number of infective centres which were established successfully following the manual inoculation of French bean leaves with tobacco necrosis virus strain D (TNV d ) or with TNV d RNA, decreased with increasing temperature between 13 and 30 °C. At 30 °C or above, primary and probably also secondary infections could not be established, though it is likely that a limited amount of virus RNA and nucleoprotein was produced at 30 °C in cells in which infection had been established previously at 23 °C. During the first day after inoculation, 23 °C was optimal for virus accumulation. Between 23 and 30 °C the rate at which lesions increased in diameter decreased with increasing temperature. The inhibitory effect of supraoptimal temperatures on the establishment of infection may be due to degradation of the infective entity by ribonuclease(s).