Cymbidium Mosaic Virus Infection Research Articles

Cymbidium Mosaic Virus Infecting Orchids: What, How, and What Next?

Context: Cymbidium mosaic virus (CymMV) is one of the most devastating viruses causing losses in the orchid industry, affecting economies worth millions of US dollars. CymMV significantly affects the orchid population and could be controlled through an integrated management strategy consisting of virus detection, good sanitation care of gardeners and their tools, and maintaining virus-free explants.Evidence acquisition: This review was written based on research publications relevant to the CymMV infection in orchids. The literature cited were obtained from online literature databases such as web of Science, Scopus, and Google Scholar. The searched term used was “Cymbidium mosaic virus”. Related publications to the initial search were also examined.Results & Conclusions: This review describes the threat of CymMV to the orchid population by examining its history, genome organization, symptoms on individual orchids, detection, and management. Current research has been focusing on the prospect of transgenic orchids with viral resistance. This review also highlights the potential role of the symbiotic relationship between orchids and arbuscular mycorrhiza fungi that could be useful to improve the protection of orchids against virus infection. Overall, this review provides information on how CymMV infection impacts the orchid population.

Identification and Characterization of NPR1 and PR1 Homologs in Cymbidium orchids in Response to Multiple Hormones, Salinity and Viral Stresses.

The plant nonexpressor of pathogenesis-related 1 (NPR1) and pathogenesis-associated 1 (PR1) genes play fundamental roles in plant immunity response, as well as abiotic-stress tolerance. Nevertheless, comprehensive identification and characterization of NPR1 and PR1 homologs has not been conducted to date in Cymbidium orchids, a valuable industrial crop cultivated as ornamental and medicinal plants worldwide. Herein, three NPR1-like (referred to as CsNPR1-1, CsNPR1-2, and CsNPR1-3) and two PR1-like (CsPR1-1 and CsPR1-2) genes were genome-widely identified from Cymbidium orchids. Sequence and phylogenetic analysis revealed that CsNPR1-1 and CsNPR1-2 were grouped closest to NPR1 homologs in Zea mays (sharing 81.98% identity) and Phalaenopsis (64.14%), while CsNPR1-3 was classified into a distinct group with Oryza sativa NPR 3 (57.72%). CsPR1-1 and CsPR1-2 were both grouped closest to Phalaenopsis PR1 and other monocot plants. Expression profiling showed that CsNPR1 and CsPR1 were highly expressed in stem/pseudobulb and/or flower. Salicylic acid (SA) and hydrogen peroxide (H2O2) significantly up-regulated expressions of CsNPR1-2, CsPR1-1 and CsPR1-2, while CsNPR1-3, CsPR1-1 and CsPR1-2 were significantly up-regulated by abscisic acid (ABA) or salinity (NaCl) stress. In vitro transcripts of entire Cymbidium mosaic virus (CymMV) genomic RNA were successfully transfected into Cymbidium protoplasts, and the CymMV infection up-regulated the expression of CsNPR1-2, CsPR1-1 and CsPR1-2. Additionally, these genes were transiently expressed in Cymbidium protoplasts for subcellular localization analysis, and the presence of SA led to the nuclear translocation of the CsNPR1-2 protein, and the transient expression of CsNPR1-2 greatly enhanced the expression of CsPR1-1 and CsPR1-2. Collectively, the CsNPR1-2-mediated signaling pathway is SA-dependent, and confers to the defense against CymMV infection in Cymbidium orchids.

Survey of the Incidence of Viral Infections in Calanthe spp. and Characterization of a GW Isolate of Cymbidium mosaic virus in Korea

Cymbidium mosaic virus (CymMV) is a major virus infecting orchid plants and causing economic loss. In this study, the incidence of viral infection in Calanthe spp. at the Korean Institute of Calanthe was investigated using reverse transcription polymerase chain reaction. The CymMV infection rate was 42%, and the two viruses Odontoglossum ringspot virus and Cucumber mosaic virus had frequencies of 8% and 2%, respectively. Additionally, we characterized an isolate of CymMV, CymMV-GW, using biological tests and examined the nucleotide sequence properties of its complete genome. CymMVGW induced chlorotic ringspots and chlorotic spot symptoms in inoculated leaves of Chenopodium amaranticolor and Nicotiana benthamiana, respectively. In this study, we have for the first complete genome sequence of CymMV-GW in Korea. The CymMV-GW genome was 6,225 nucleotides in length, excluding the poly-(A) tail, and showed whole-genome nucleotide and amino acid sequence identities of 97.7% and 100%, respectively, with the NJ-1 isolate of CymMV. Here, we report the complete genome sequence of the CymMV-GW isolate and viral infection rates for Calanthe spp. in Korea.

Differentially expressed genes of Chenopodium amaranticolor in response to cymbidium mosaic virus infection

Cymbidium mosaic virus (CymMV)-induced expressed sequence tag (EST) clones from Chenopodium amaranticolor were identified. CymMV was mechanically inoculated onto C. amaranticolor, and local lesion symptoms were observed. Inoculated leaves were collected on serial days post inoculation (dpi) to identify activated or suppressed genes. mRNA isolation and suppression subtractive hybridization (SSH) were then performed to identify differentially expressed genes related to the local lesion response. Fifty-three ESTs, including genes related to defense and stress responses (e.g., lipoxygenase, jasmonate-induced protein, and heat shock protein), were generated. In addition, a large proportion of the ESTs were found to be involved in photosynthesis, as determined by their functional categories. Expression levels of several EST genes were observed using quantitative real-time reverse transcription-polymerase chain reaction, and the evaluated genes showed varying levels of expression during the experimental period. In this study, differentially expressed sequences via SSH were identified from CymMV-infected C. amaranticolor, and profiling and annotation were carried out to determine the expression pattern of CymMV and its interaction with C. amaranticolor.

The Bias in Small RNA Profiles Between Symptomless Dendrobium and Severe Symptom Ascocenda Orchids Infected Long-Term with Cymbidium Mosaic Virus

Perennial orchids worldwide are frequently infected with Cymbidium mosaic virus (CymMV). To date, no studies have described non-coding small RNA (sRNA) responses in the CymMV long-term-infected orchids. In this study, sRNA profiles of microRNAs (miRNA) and small virus-derived interfering RNAs (siRNA) in symptomless, long-term CymMV-infected Dendrobium and severe symptom, long-term CymMV-infected Ascocenda were identified in four sRNA libraries of virus-free and CymMV-infected orchids by Illumina high-throughput sequencing. The miRNA analysis revealed that the infected orchids had unique sRNA profiles that differed in size distribution, specially the presence of 21 nt sequences in severe symptom Ascocenda. Gel blot analysis showed a change in the miR528 expression which was at high level in virus free Ascocenda, on the other hand, only detected at low level in severe symptom Ascocenda. This expression alteration was also detected with the miR156 expression level in Dendrobium. Moreover, the characterization in polarity of CymMV-derived siRNAs in both infected orchids demonstrated that antisense polarity siRNAs accumulated in severe symptom Ascocenda, and the symptomless Dendrobium contained more sense polarity siRNAs. Here, we showed that miRNA expression was altered after long-term CymMV infection. Furthermore, a different pattern of siRNAs was observed between two orchid genera infected with the same virus and depended on the symptom severity.

The NPR1 ortholog PhaNPR1 is required for the induction of PhaPR1 in Phalaenopsis aphrodite.

BackgroundSystematic acquired resistance (SAR) is an effective broad-spectrum defense mechanism that confers long-lasting protection against biotrophic pathogens trough defense related salicylic acid (SA) signaling. Gene(s) involved in SAR have been extensively studied in dicot plants; however, remains largely unresolved in monocot plants. NPR1, an evolutionary conserved gene, plays a central role in SAR, and PR-1 is widely used as a marker for effective SA signaling.ResultsWe identified NPR1 and PR-1 homologous genes, PhaNPR1 and PhaPR1, from an economically important orchid, Phalaenopsis aphrodite, and characterized their roles in SA signaling and Cymbidium mosaic virus (CymMV) resistance. A phylogenetic analysis of NPR1 homologs showed that these genes appear to have evolved before angiospermy. Similar to Arabidopsis NPR1, PhaNPR1 was only moderately induced upon SA treatment and CymMV infection. Although PhaPR1 shows only 36% identity with AtPR1, its promoter shared conserved elements with those of other PR-1 genes, and it was induced upon SA treatment and CymMV infection. After CymMV infection, silencing on PhaNPR1 also reduced PhaPR1 expression; however, CymMV accumulation was not affected.ConclusionsIn conclusion, after virus infection, PhaNPR1 is required for PhaPR1 induction, but plays little role in defense against CymMV.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-31) contains supplementary material, which is available to authorized users.

Incidence of Cymbidium mosaic virus and Odontoglossum ringspot virus in Dendrobium spp. in Thailand

Dendrobium is the most popular cut flower and potted plant for the Thai orchid industry and several viruses have been reported to negatively impact upon their growth and yield. This study was carried out to confirm viral infection and identify the viruses infecting Dendrobium spp. in Thailand. Dendrobium orchids propagated through cutting of pseudobulbs, and grown in tissue culture were screened for Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) with indirect enzyme-linked immunosorbent assay (ELISA). CymMV and ORSV were detected in 65.4% and 0% of the 280 tested plants, respectively. Among the 29 cultivars of tissue cultured cut flower and 19 cultivars of potted Dendrobium, CymMV infection rates were 18.6% and 8.7%, respectively. No CymMV or ORSV were found in 21 species of in vitro seed cultured seedlings of Thai native Dendrobium. The data indicated that CymMV is a prevalent virus in vegetatively propagated Dendrobium but not in in vitro seed cultured seedlings in Thailand. Plant material must be tested for the presence of viruses before being used for mass production by tissue culture, and sanitation during propagation of orchid plants and harvesting of flowers is essential in order to prevent the virus from spreading.

Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium Mosaic Virus is a manifestation of RNA-mediated resistance

Phalaenopsis orchid is grown extensively for commercial production as cut flowers and potted plants. Susceptibility to the cymbidium mosaic virus (CymMV) has drawn concern among orchid growers worldwide as its natural host remains confined to orchids. Hence it was reasoned to improve the resistance of Phalaenopsis to CymMV by expressing a virus-derived transgene. A DNA cassette containing a CymMV coat protein (CP) cDNA and a nos terminator placed downstream of a maize ubiquitin promoter was transformed into Phalaenopsis orchid by particle bombardment. Southern blot analysis of transgenic orchid plants was conducted to confirm the successful integration of multiple copies of the CP gene in the orchid genome. Although low levels of CP mRNA transcript signals were observed by northern blot analysis, CP was not detected by western blot analysis in transgenic orchid plants. However these transgenic orchid plants exhibited enhanced resistance to virus infection, as confirmed by RT-PCR and ELISA. T0 progeny lines were assayed for resistance to CymMV as it occurs in the field. Five among the 13 tested lines showed CymMV protection in more than 50% of their progeny. Nuclear run-on and small interfering RNA (siRNAs) analyses showed that CymMV resistance was RNA-mediated through a post-transcriptional gene silencing mechanism (PTGS) in the silenced transgenic orchid plants. The detection of siRNAs, and not CP mRNA, proved to be an indicator of their resistance to CymMV infection. Results from the current study indicate that Phalaenopsis expressing heterologous CymMV CP can effectively confer resistance to CymMV.

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants.

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3' end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23,600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WClMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.

Gene products encoded by cymbidium mosaic virus RNA: proteins translated in vitro

The potexvirus, cymbidium mosaic virus (CyMV), a major pathogen of cultivated orchids, has a single-stranded RNA genome that serves, all or in part, as messenger for translation of viral proteins during infection. As a first step in investigating the mechanisms of gene expression in CyMV infection, we have focused on the question of the protein-encoding potential of the viral RNA. CyMV particles have been highly purified from infected Datura stramonium leaves using Percoll gradient sedimentation and intact RNA has been extracted from the virus and used as messenger in an in vitro translation system from wheat germ. Translated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. CyMV genomic RNA encoded at least eight major polypeptide species in the in vitro translation system. Our results suggest that some protein processing by specific cleavage might occur as part of the process of gene expression.