Odontoglossum Ringspot Tobamovirus Research Articles

Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus

Taxonomically distinct Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are two of the most prevalent viruses worldwide; when co-infecting orchids, they cause synergistic symptoms. Because of the huge economic loss in quality and quantity in the orchid industry with virus-infected orchids, virus-resistant orchids are urgently needed. To date, no transgenic resistant lines against these two viruses have been reported. In this study, we generated transgenic Nicotiana benthamiana expressing various constructs of partial CymMV and ORSV genomes. Several transgenic lines grew normally and remained symptomless after mixed inoculation with CymMV and ORSV. The replication of CymMV and ORSV was approximately 70–90% lower in protoplasts of transgenic lines than wild-type (WT) plants. Of note, we detected extremely low or no viral RNA or capsid protein of CymMV and ORSV in systemic leaves of transgenic lines after co-infection. Grafting experiments further revealed that CymMV and ORSV trafficked extremely inefficiently from co-infected WT stocks to transgenic scions, presumably due to RNA-mediated interference. This study reports the first successful creation of dual resistant transgenic lines against CymMV and ORSV. Our studies shed light on the commercial development of transgenic orchid production to combat the global viral threat.

Characterization and application of a common epitope recognized by a broad-spectrum C4 monoclonal antibody against capsid proteins of plant potyviruses.

A broad-spectrum monoclonal antibody (C4 MAb) against the capsid proteins (CPs) of plant potyviruses has been generated in previous studies. To clarify which epitope is recognized by this MAb, epitope mapping was performed via phage display library screening and amino acid substitution analysis. Subsequently, a 12-residue epitope in the core region of potyvirus CPs was identified and termed the C4 epitope (WxMMDGxxQxxY/F). This epitope contains tryptophan and tyrosine residues that are crucial for reacting with C4 MAb. The CP of Odontoglossum ringspot tobamovirus (ORSV) separately fused with the C4 epitope of Konjak mosaic potyvirus (KoMV), Zantedeschia mild mosaic potyvirus (ZaMMV), or Dasheen mosaic potyvirus (DsMV) was expressed in a bacterial system and purified. The results of indirect ELISA and Western blotting demonstrated that the C4 epitope of KoMV (Ko) fused to ORSV CP showed the strongest binding affinity to C4 MAb among the three viral epitope tags examined. The binding affinity between Ko tag (WTMMDGEEQIEY) and C4 MAb was determined. To examine the applicability of the Ko tag in planta, GFP and ORSV CP were transiently expressed in Nicotiana benthamiana, and both Ko-tagged proteins were specifically detected using C4 MAb. The Ko tag did not affect the silencing suppressor function of Tomato bushy stunt tombusvirus P19 in N. benthamiana. Furthermore, Ko-tagged EGFP could be successfully expressed, specifically detected and subsequently immunoprecipitated using C4 MAb in a mammalian cell system. Thus, the present study identified a common C4 epitope of potyviruses recognized by the broad-spectrum C4 and PTY 1 MAbs, and the results indicated that the newly designed Ko tag is suitable for application in bacterial, plant, and mammalian cell systems.

Cloning and sequence analysis of orchids CymMV and ORSV CP gene isolated in northern of Vietnam

Orchids are ones of indoor decorated plants with beautiful and fragrant flowers. It is not only varied in styles, but also high economic valuable flower. However, the cultivation of orchids meets several difficulties, especially viral infection that caused reducing the productivity and quality of orchid flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot tobamo virus (ORSV) are considered as viruses that cause the largest damage to orchids. In this study, we cloned and sequenced the coating protein genes (CP) of the two viruses CymMV and ORSV isolated from some provinces in northern Vietnam. These sequences were aligned out by BioEdit 7 software. The CP nucleotide sequences of the isolates were compared with those of previously published isolates in GenBank originating from different countries. The results showed the CP gene sequences of CymMV were quite diverse, in contrast, the CP genes of ORSV were very conservative. These results contributed to the study of genetic diversity of the CymMV and ORSV viruses and as a basis for selection genetic materials to create disease resistant orchids in Vietnam.

Development of a sensitive diagnostic assay to detect Cymbidium mosaic virus and Odontoglossum ringspot virus in members of the Orchidaceae

SummaryThe Orchidaceae is a family of flowering plants native to China, members of which possess high ornamental and pharmacological value. Many members of the family Orchidaceae are susceptible to infection by both Odontoglossum ringspot tobamovirus (ORSV) and Cymbidium mosaic potexvirus (CymMV). Symptoms of viral infection include ringspots, mosaics, mottles, chlorotic streaks, and stripes on the leaves, and viral infections cause significant losses in the commercial value of these plants. However, there are no effective measures to control ORSV and CymMV. Therefore, it is vital to identify viral epidemics through the rapid and efficient detection of these two viruses, in order to limit their economic damage. We have developed an assay for detecting co-infection of members of the Orchidaceae by ORSV and CymMV. The coat protein (CP) genes from ORSV and CymMV were cloned and the CPs were expressed in vitro and used to generate two virus-specific polyclonal antisera that were validated by western blotting. Polyclonal anti-ORSV-CP and anti-CymMV-CP antisera were used simultaneously to develop a bi-immunocapture reverse transcription-polymerase chain reaction (BIC RT-PCR) assay. We demonstrated that the BIC RT-PCR assay was sensitive and specific, and provided a valuable tool for the early detection of ORSV and CymMV in leaf sap extracts from plants of the Orchidaceae.

Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors

Quartz crystal microbalance (QCM) immunosensors are based on the principle that adsorption of substances on the surface of a quartz crystal changes its resonance oscillation frequency. A QCM immunosensor was developed for the detection of both cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) by pre-coating the QCMs with virus-specific antibodies. Upon binding of virions in either purified form or crude sap of infected orchids with the immobilised virus antibodies, the increase in mass at the QCM surface resulted in a reduction in the frequency of resonance oscillation in a manner dependent upon the amount of virus bound. The QCM was able to detect as low as 1 ng each of the two orchid viruses. This detection sensitivity is comparable to enzyme linked immunosorbent assay (ELISA) but the assay is faster. This immunoassay was shown to be specific, sensitive, rapid and economical, thus providing a viable alternative to virus detection methods. This is the first report using QCM immunosensors to detect plant viruses.

Simultaneous quantitation of two orchid viruses by the TaqMan ® real-time RT-PCR

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan ® real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan ® probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5′ terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3′ terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle ( C T) values were determined and a linear relationship was established between these C T values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 10 4 copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

Rapid simultaneous detection of two orchid viruses using LC- and/or MALDI-mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry are capable of providing molecular mass information on biological samples with high speed, accuracy and sensitivity. With mass spectrometry, identifying a virus based on the molecular weight of its coat protein is relatively simple and accurate. The technique can be applied to all viruses with known coat protein molecular weights. Using the LC/MS and/or MALDI, this paper describes rapid simultaneous detection of the two most prevalent orchid viruses, namely cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV). The coat protein molecular weights of CymMV and ORSV were detected accurately using an extract from 1 g of virus-infected Oncidium orchid flower. Because LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps, both techniques are ideal for rapid identification of viruses from a large number of samples. This is the first report on the application of LC/MS and/or MALDI for simultaneous detection of two plant viruses from an infected plant extract.

Detection of Cymbidium Mosaic Potexvirus and Odontoglossum Ringspot Tobamovirus Using Immuno-Capillary Zone Electrophoresis

ABSTRACT Immuno-capillary zone electrophoresis (I-CZE) is a technique that combines the specificity afforded by serological assays with the sensitivity, rapidity, and automation in detection provided by capillary zone electrophoresis. Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) were detected in their purified forms as well as in the crude saps of infected Nicotiana benthamiana leaves and Oncidium orchid flowers. The two orchid virus-antibody complexes were resolved via the combined actions of electrophoretic migration and electro-osmotic flow along a buffer-filled, uncoated fused-silica capillary. The I-CZE fractions collected from both CymMV- and ORSV-antibody complex peaks, as well as the RNA purified from them, retained their infectivity upon inoculation onto Chenopodium quinoa. I-CZE assays were able to detect as little as 10 fg each of both CymMV and ORSV in their purified forms as well as in the crude saps of infected N. benthamiana and Oncidium orchid. As multiple samples can be analyzed rapidly, I-CZE offers an ideal diagnostic technique for routine mass-indexing programs such as virus-free certification, breeding for virus-resistant cultivars, plant quarantine, and germ plasm screening. This is the first report of the application of I-CZE for the detection of plant viruses.

The GPRIME package: computer programs for identifying the best regions of aligned genes to target in nucleic acid hybridisation-based diagnostic tests, and their use with plant viruses

The GPRIME (Group PRIMEr design) programs examine aligned sets of gene sequences to discover homologous regions to be targeted in diagnostic tests. The core program moves a `window' over the aligned sequences and calculates, at each window position, a `redundancy value', namely the number of sequences that would represent all permutations of the variable sequence positions within that window. Regions with minimal redundancy values may then be targeted in diagnostic tests based on oligonucleotide hybridisation. The likely specificity of tests targeting such regions can be assessed by searching the international databases with those regions using FASTA. The GPRIME programs, which include programs for designing primers to distinguish between two sub-sets of a group of aligned sequences, can be obtained from http://life.anu.edu.au/software.html. We have used GPRIME to design redundant primers for RT-PCR tests to detect all potexviruses and tobamoviruses, and then used these, together with a previously reported pair of primers for the Potyviridae, to screen some Australian orchid collections. Two orchid viruses previously reported from Australia were found; cymbidium mosaic potexvirus was common, but odontoglossum ringspot tobamovirus was not. In addition the recently described ceratobium mosaic potyvirus was found to be common, and three other novel potyviruses were also found.

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts.

An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 microgram and 4 micrograms per 2 x 10(6) protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the "Replication Footprint Profiles" (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts.

Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers

Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) are the two most prevalent and economically important orchid viruses which have attained a world-wide distribution. We have designed one pair of common primers, which were able to detect, simultaneously, both viruses by a single polymerase chain reaction (PCR). Sequence alignment and primer analyses were employed in primer design. The effect of touch-down PCR, primer annealing temperature, number of thermal cycles, MgCl 2 concentration and the degree of primer degeneracy, were investigated and applied to the optimisation of PCR conditions in the detection of both viruses in order to achieve equal amplification efficiencies. This is the first successful attempt at using a single pair of PCR primers to detect two viruses belonging to different taxonomic groups simultaneously.

The use of DIG-labelled cRNA probes for the detection of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) in orchids

DIG-labelled sense and antisense cRNA probes were synthesized from cDNA clones of CymMV and ORSV for virus detection in infected plants. A slot-blot hybridization assay was developed using either crude leaf extracts or total RNA from infected leaves. The assay could detect 50 and 250 pg of purified CymMV and ORSV RNA, respectively. As little as 30 mg of Nicotiana benthamiana infected leaves was sufficient to provide positive detection. CymMV and ORSV were detected at 3125 and 625 times dilution of leaf extracts, respectively. The DIG-labelled cRNA probes are stable for more than a year. This method is sensitive, reliable and suitable for large-scale routine testing of plant viruses. By using the two DIG-labelled cRNA probes in situ, CymMV and ORSV were localized in systemically infected leaves and stems of N. benthamiana and orchids.

Diagnostic PCR between dream and reality — The clinicians' view

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan® real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan® probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5′ terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3′ terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle (CT) values were determined and a linear relationship was established between these CT values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 104 copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

Detection of Cymbidium Mosaic Potexvirus and Odontoglossum Ringspot Tobamovirus from Thai Orchids by Rapid Immunofilter Paper Assay.

During 1992 to 1994, 442 orchid plants from 24 genera in 43 nurseries and locations in eight provinces of northern, central, and southern regions of Thailand were examined for Cymbidium mosaic potexvirus (CyMV) and Odontoglossum ringspot tobamovirus (ORSV) with rapid immunofilter paper assay (RIPA). Both viruses were confirmed in all three regions. However, CyMV was detected in 17 genera in 93% nurseries, while ORSV was detected in only four genera (Arachnis, Cattleya, Oncidium, and Vanda) in 40% of nurseries tested. These results indicate that CyMV is more prevalent than ORSV in Thailand. Mixed infections of ORSV and CyMV were often found, and double infections were observed in 95% of ORSV-infected plants. The virus infections were divided into two categories: infection by CyMV alone and double infection by both viruses in Cattleya and Oncidium. CyMV was often detected alone from symptomatic or asymptomatic plants of certain orchid genera. In Oncidium, however, CyMV was detected alone from 79% of plants without symptoms, while both viruses were detected from 90% of plants with mosaic and necrotic spot symptoms on leaves. Expression of symptoms may be promoted by the double infection of CyMV and ORSV in Oncidium.

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications.

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.

An appraisal of the banded and paracrystalline cytoplasmic inclusions induced in cymbidium mosaic potexvirus- and odontoglossum ringspot tobamovirus infected orchid cells using confocal laser scanning microscropy.

The cymbidium mosaic potexvirus (CyMV) banded inclusions and the odontoglossum ringspot tobamovirus (ORSV) paracrystalline inclusions were studied in flowers and leaves of nine orchid cultivars using the confocal laser scanning microscope. The inclusions varied in length and width and were mostly located adjacent to the cell walls. Some ORSV inclusions fully extended across entire infected cells. We propose that the CyMV banded inclusion was formed from virus aggregates which aligned as periodical stacked layers, appearing as cross bands. The virus aggregates were folded into flexible inclusions, giving rise to various shapes and forms. The ORSV paracrystalline inclusions were observed as needle-like crystals. The confocal laser scanning microscope is an effective tool for the study of the three-dimensional structures of plant virus induced inclusions.

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification.

The odontoglossum ringspot Tobamovirus (ORSV) movement and coat proteins genes were selected for the design of oligonucleotide primers for amplification of a 1,085 bp fragment. A combined assay of reverse transcription and the polymerase chain reaction (RT-PCR) was performed with 20-mer ORSV-specific primers and crude nucleic acid extracts from virus-infected orchids for rapid detection of the virus. The lowest concentration of template viral RNA required for detection was 10 fg. The RT-PCR is a 10(3) times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. No PCR product was observed when cymbidium mosaic potexvirus or a crude extract of healthy Cymbidium sp. were used as a template in RT-PCR with the same primers. The specificity of the primers was verified using other tobamoviruses RNAs.

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).

Cloning of the 3'-terminal region encoding movement and coat proteins of a Korean isolate of odontoglossum ringspot virus.

The 3'-terminal 1855 nucleotides (nts) of a Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) were cloned and sequenced. The sequence contained two open reading frames, which encode the cell-to-cell movement protein (MP) and coat protein genes, and are 912 nts and 477 nts long, respectively. The MP gene contained a conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA locating between 1117 nts and 1292 nts from the 3'-end. The 3' untranslated region (UTR) of the virus comprises 414 nts, includes nine pseudoknots and a tRNA-like structure domain containing aminoacyl acceptor arm and the anticodon hairpin loop coding for histidine.

Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant.

Tobacco mosaic virus (TMV) which contains the movement protein (MP) of odontoglossum ringspot tobamovirus (ORSV) in place of the TMV MP systemically infects orchids but causes local infection in tobacco unless the carboxy-terminal 48 amino acids of the MP are deleted (C. A. Holt, C. A. Fenczik, S. J. Casper, and R. N. Beachy; Virology, in press, 1995). Frameshift mutations were created within the 3' ends of the MP gene that led to truncations of the ORSV MP by 11, 19, 28, 37, and 48 amino acids; each of the mutant MP genes was inserted into the cloned cDNA of TMV in place of the TMV MP and infectious transcripts were produced. Virus containing mutant MPs were used to infect vanilla orchids, a systemic host of ORSV, and tobacco plants. Removal of 11 amino acids from the ORSV MP prevented spread of the chimeric virus in orchids while restoring the ability to cause a systemic infection on tobacco. Further deletions of the MP affected the size of virus-induced necrotic local lesions on tobacco cv. Xanthi NN and the systemic spread and accumulation of virus in cv. Xanthi nn, a systemic host of TMV. However, each virus replicated to equivalent levels in protoplasts. A mechanism by which the ORSV MP limits the spread of the chimeric virus is proposed.