Turnip Yellow Mosaic Tymovirus Research Articles

Diversity of Ubiquitin and ISG15 Specificity among Nairoviruses' Viral Ovarian Tumor Domain Proteases

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

Identification and functional analysis of the turnip yellow mosaic tymovirus subgenomic promoter.

Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.

An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method

An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2×10 4 infected protoplasts.

Effects of turnip mosaic virus and turnip yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea)

SUMMARYWild plants of Brassica oleracea (wild cabbage) are commonly infected with turnip mosaic poty virus (TuMV), turnip yellow mosaic tymovirus (TYMV) and several other viruses. A field experiment in which plants were inoculated either with TuMV or TYMV showed that virus infection significantly reduced survival, growth and reproduction. Relative to water inoculated‐controls, plants infected with TYMV had greater mortality, were shorter, had a smaller leaf area and number, showed a greater amount of damage from herbivory and chlorosis, were less likely to flower and produced fewer pods and lower total seed output. Plants infected with TuMV did not appear to be adversely affected at first; however, mortality after 18 months was higher than control plants. Although TuMV infection had no effect on the number of plants flowering, the infected plants did produce fewer pods and a lower total seed output. We conclude that both viruses can significantly affect vegetative and reproductive performance of wild cabbage and hence that introgression of virus resistance (particularly when conferred by a major gene or a transgene) from a crop might increase plant fitness in natural populations of this species. Ecological risk assessments of virus resistance transgenes must do more than survey adult plants in natural populations for the presence of the target virus. Failure to detect the virus could be due to high mortality on infection with the virus.

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea.

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.

Virion protein sequence variation among Australian isolates of turnip yellow mosaic tymovirus.

The virion protein genes, and 3' untranslated regions, of six variants of turnip yellow mosaic tymovirus (TYMV) that produced different symptoms in their native host Cardamine robusta and in Chinese cabbage plants, have been sequenced. The sequences have been compared with each other, and with the same region of the pBL-16 clone of the Blue Lake isolate of TYMV. The sequences of the virion protein genes differed by a mean of 1.89% (range 0-2.82%), and the encoded proteins by a mean of 1.71% (range 0-3.17%). The nucleotide differences were confined to the 5'-most 60% of the gene, whereas there were amino acid differences only among residues 12 to 29 and residue 102 (numbered from the N-terminus) of the virion protein involving only hydrophobic residues at the surface of the protein. The amino acid and nucleotide differences between the seven isolates did not correlate with differences in the symptoms they caused, but confirmed earlier estimates of genetic variability in the wild populations of the virus.

RAPD variation in populations of Cardamine lilacina (Brassicaceae)

Random amplified polymorphic DNA (RAPD) analysis of DNA samples clearly distinguished between Cardantine hirsuta L. and two variants of Cardamine lilacina Hook. found in Australia. Cardarnine hirsuta, a recent immigrant, was less variable than C. lilacina, robust alpine snow-patch variant (C. lilacina-RASPV), which is an endemic plant that grows in three distinct regions in the Kosciusko alpine area and C. lilacina, intermedia variant (C. lilacina-IV), which is an endemic plant that is widespread in the same area. Comparison of RAPDs showed that, in the populations of C. lilacina-RASPV, some plants found near each other were closely related, but there was no detectable largerscale population structure. It is most likely that the present populations of C. lilacina-RASPV are relics of a single larger heterogeneous population. Cardarnine lilacina-RASPV is the only known natural host of turnip yellow mosaic tymovirus in Australia. Although in this study RAPD analysis did not indicate whether the host plant variation is influencing the variability of populations of this virus it did provide an understanding of the genetic relationships in these populations of C. lilacina.

Genetic variation of turnip yellow mosaic tymovirus within single plants of Cardamine lilacina

An RNA hybrid mismatch polymorphism (RHMP) method was used to investigate genomic variation of turnip yellow mosaic tymovirus (TYMV) within single plants of its wild perennial host Cardamine lilacina. Two probes covering portions of the three TYMV genes were used to examine the viral RNA isolated directly from single shoots of the host plant. Up to 16 separate shoots of each infected plant, including some which were attached to others by their perennial rhizomatous bases, were analysed. The RHMP results demonstrated that TYMV often varied within single plants, and even within single shoots, in both regions of the genome tested. More within-plant viral variation was observed when shoots were collected from sites with many infected plants than from sites where few plants were infected, suggesting that proximity to other infected plants may facilitate coinfection and recombination between TYMV isolates.

Expression of the turnip yellow mosaic virus proteinase in Escherichia coli and determination of the cleavage site within the 206 kDa protein.

The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C-terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.

RNA hybrid mismatch polymorphisms in Australian populations of turnip yellow mosaic tymovirus.

In the Mt. Kosciusko alpine area of Australia there are three well-separated populations of Cardamine lilacina, an endemic sward-forming perennial brassica, and these are infected with turnip yellow mosaic tymovirus. The genetic variation in these viral populations has been assessed by an RNA hybrid mismatch polymorphism method. About 100 isolates were examined; the genomic RNA of each isolate was prepared from a shoot of a single wild C. lilacina plant. RNA hybrid mismatch polymorphisms (RHMPs) were assessed in six regions of the genomes using labelled negative-strand probes transcribed from selected portions of a cloned TYMV genome. The probed region at the 3' end of the genome showed little variation and over 95% of the isolates gave the same pattern. However, other parts of the genome, including the 5' non-coding region, were much more variable. There was no significant correlation between groupings based on the RHMP patterns, and the location from which the isolates were collected, nor with the symptom type or severity shown by their host plants. The patterns of variation suggested that all three populations of the virus are a single quasi-species; at most one tenth of the isolates gave similar RHMP patterns, those of the "master copy".

Infectious eggplant mosaic tymovirus and ononis yellow mosaic tymovirus from cloned cDNA.

Eggplant mosaic virus (EMV) and ononis yellow mosaic virus (OYMV) are two tymoviruses that have ssRNA genomes of about 6.2 kb and 6.3 kb, and which infect solanaceous and leguminous hosts, respectively. Full-length cDNA clones of these viruses were constructed with a T7 promoter adjacent to the 5' terminus of the DNA copy of the viral genome, and with unique restriction endonuclease sites at the 3' terminus. This allowed RNA to be transcribed from the DNA encoding the genome. The transcript RNA was infectious when inoculated to Nicotiana glutinosa (for EMV) and Pisum sativum (for OYMV). These clones, together with clones of turnip yellow mosaic tymovirus, which infects brassicas, have been used to construct hybrids in which the virion protein gene was exchanged between EMV or OYMV and turnip yellow mosaic virus. These and other hybrids are being used to investigate the molecular basis for host range differences in tymoviruses.

Mosaic disease induced by turnip yellow mosaic Tymovirus

Mosaic disease is one of the most common consequences of infection of plants by viruses. Most such mosaics include islands of dark green tissue, which are normal in appearance, contain little or no virus and are resistant to superinfection at least for a period of weeks. The mosaic pattern that develops in successive expanded leaves of larger plants is variable and unpredictable. Therefore it has not been possible to investigate the early events in leaf primordia that lead to the formation of dark green tissue. Here we describe an experimental system which should allow the application of molecular techniques to the study of the nature of dark green tissue.

Nucleotide sequence of the genome of eggplant mosaic tymovirus

The sequence of the RNA genome of an isolate of eggplant mosaic tymovirus from Trinidad (EMV-Trin) has been determined. The genome is 6330 nucleotide residues in length and contains three open reading frames; two overlapping genes, whose initiation codons are separated by seven nucleotide residues (nucleotide residues 102–2051 and 109–5628) near the 5′ terminus, and the virion protein gene, which is near the 3′ terminus (nucleotide residues 5633–6199). The genomes of EMV-Trin and turnip yellow mosaic tymovirus have the same genomic organization and similar nucleotide and encoded amino acid sequences. The nucleotide residues adjacent to the initiation codons of tymoviral overlapping genes have closely similar sequences which may form a weak stem-loop secondary structure that regulates their translation.

Nucleotide sequence of the genome of an Australian isolate of turnip yellow mosaic tymovirus

The nucleotide sequence of the Club Lake isolate of turnip yellow mosaic virus (TYMV-CL) genomic RNA has been determined. The genome is 6319 nucleotide residues in length and has three major open reading frames (ORFs), two of which overlap. The smallest ORF is proximal to the 3' terminus and encodes the virion protein gene, which has 98% sequence similarity with the virion protein gene reported for the type strain of TYMV. The largest ORF is from nucleotide residues 96 to 5630, and encodes a protein some parts of which show sequence similarities to the possible RNA replicases and nucleotide binding proteins of other viruses. The third ORF is from nucleotide residues 89 to 1975 and overlaps the 5' end of the largest ORF in a manner similar to that found in several animal viral genomes. The function of the protein encoded by this ORF is unknown. The genomes of tymoviruses have, characteristically, an unusually large cytosine content and small guanosine content. This compositional bias is mirrored in the codon and dinucleotide frequencies of the TYMV-CL genome, but is only partially reflected in the amino acid sequences encoded by the genome.

Further studies on turnip yellow mosaic tymovirus isolates from an endemic Australian Cardamine

Native and introduced species of Cardamine and other brassicas were collected from various parts of south‐eastern Australia and tested for sap‐transmitted viruses. Isolates of turnip yellow mosaic virus (TYMV‐Cd) were obtained from a robust sward‐forming (SF) species, C. lilacina, that is an endemic species and restricted to the high glacial cirques of the Kosciusko alpine area. At two sites (Blue Lake and Club Lake) 22% of the plants were infected. An undescribed species of flightless pill beetle, Pedilophorus (Byrrhidae), was found on the C. lilacina SF plants. They preferred feeding on leaf discs infected with TYMV‐Cd rather than on virus‐free leaf discs and transmitted the virus for 48 h to seedlings of C. lilacina SF (2.5%) or Chinese cabbage (10%).The pattern of distribution of TYMV‐Cd and its close association with Pedilophorus suggested that it is not a recent migrant to the area. The possible time of its arrival is discussed.A carlavirus was isolated from up to 4% of three other native highland Cardamine species.

Effect of viruses on pollen morphology

Many pollen grains from Chenopodium quinoa plants infected with sowhane mosaic sobemovirus (SMV) were collapsed, grooved and had sunken opercula, whereas those from the first flowers of virus‐free plants were smooth, rounded and with protuberant opercula. However, pollen grains from later flowers of virus‐free plants were similar in appearance to those from the virus‐infected plants. Similar but less obvious symptoms were found in pollen of Plantago lanccoiata infected with either ribgrass mosaic tobamovirus or broad bean wilt virus. No symptoms were found in pollen of Hordeum vulgare cv. clipper, H. sponiaitcum or Triticum acstivum infected with barley suripe mosaic hordeivirus, nor in pollen of a Cardaminc sp. naturally infected with a strain of turnip yellow mosaic tymovirus. The symptoms, even those shown by pollen from SMV‐infected C quinoa, seemed not to be sufficiently characteristic for diagnosis of virus infection.