Moon Nam

Nucleotide sequence and genomic organization of a newly identified member of the genus Carmovirus, soybean yellow mottle mosaic virus, from soybean

The viral genome of soybean yellow mottle mosaic virus (SYMMV) from infected soybean (Glycine max) in Korea was cloned and sequenced. The complete monopartite single-stranded RNA genome of SYMMV consists of 4009 base pairs with six putative open reading frames and includes 5′- and 3′-untranslated regions of 39 and 229 nucleotides, respectively. The nucleotide and coat protein sequences of SYMMV share the highest sequence identity with those of cowpea mottle virus. Based on its genomic organization, its predicted amino acid sequence, and its phylogenetic relatedness to known carmoviruses, we report that SYMMV is a new member of the genus Carmovirus in the family Tombusviridae.

2007?2011 Characteristics of Plant Virus Infections on Crop Samples Submitted from Agricultural Places

National Institute of Horticultural and Herbal Science, Horticultural and Herbal Environmental Division,Suwon 441-707, Korea(Received on September 11, 2012; Revised on October 11, 2012; Accepted on October 12, 2012)The total number of requests and associated specimens for the diagnosis of virus infection were 573 and 2,992,respectively, on crops from agricultural places of farmers, Agricultural extension services and so forth for 5years from 2007. The total number of virus tests was 13,325. The number of species of viruses infected on thesubmitted crops was 21 in 2007, 15 in 2008, 23 in 2009, 21 in 2010 and 17 in 2011. The newly recorded viruseswere Tobacco leaf curl virus (TbLCV) in 2007, Tomato yellow leaf curl virus (TYLCV) in 2008, Impatiencenecrotic spot virus (INSV) and Radish mosaic virus (RaMV) in 2009, and Beet western yellows virus (BWYV) in2010. Forty virus species including Alfalfa mosaic virus were detected over 5 years. The ten most frequentlydetected virus species were Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato leaf curlvirus (TYLCV), Cucumber green mottle mosaic virus (CGMMV), Broad bean wilt virus 2 (BBWV2), Zucchiniyellow mosaic virus (ZYMV), Melon necrotic spot virus (MNSV), Pepper mild mottle virus (PMMoV),Watermelon mosaic virus (WMV) and Pepper mottle virus (PepMoV). The types of crops submitted fromagricultural places were 51 in total and the ten most frequently submitted crops were red pepper, tomato,paprika, watermelon, melon, rice, cucumber, corn, radish and gourd. The total request rate for the top 10crops and top 20 crops was 81.6% and 94.2%, respectively. Eight pepper infecting virus species includedCMV, and the average infection rate was 24.6% for CMV, 18.9% for PMMoV and 14.7% for TSWV. Sevenkinds of double infection were detected in pepper including BBWV2+CMV at 14.7% on average, and fourtypes of triple infection including BBWV2+CMV+PepMoV at 0.9% on average. Six virus species detected ontomato including TYLCV, and the average infection rate was 50.6% for TYLCV, 14.5% for TSWV and10.9% for Tobacco leaf curl virus (TbLCV). The mixed infection of CMV+TSWV on tomato was 3.9% onaverage and of Tomato mosaic virus (ToMV)+TYLCV was 0.4% on average. Five viruses detected onwatermelon included MNSV and the average infection rate was 37.0% for MNSV, 20.4% for CGMMV,18.1% for ZYMV and 17.8% for WMV. The mixed infection rate on watermelon was CMV+MNSV andWMV+ZYMV having an average infection rate of 0.7% and 5.0%, respectively. The average infection rateson melon were 77.6% for MNSV, 5.6% for CMV and 3.3% for WMV. Mixed infections of CMV+MNSVoccurred on melon with an average infection rate of 13.5%. Keywords : BBWV2, BWVY, CGMMV, CMV, MNSV, Plant virus, TYLCV, ZYMV

Arabidopsis TTR1 Causes LRR-Dependent Lethal Systemic Necrosis, rather than Systemic Acquired Resistance, to Tobacco Ringspot Virus

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.

Genetic Compositions of Broad bean wilt virus 2 Infecting Red Pepper in Korea.

The incidence of Broad bean wilt virus 2 (BBWV2) on red pepper was investigated using the samples obtained from 24 areas of 8 provinces in Korea. Two hundred and five samples (79%) out of 260 collected samples were found to be infected with BBWV2. While the single infection rate of BBWV2 was 21.5%, the co-infection rate of BBWV2 with Cucumber mosaic virus, Pepper mottle virus, Pepper mild mottle virus and/or Potato virus Y was 78.5%. To characterize the genetic diversity of BBWV2 Korean isolates, 7 isolates were fully sequenced and analyzed. Phylogenetic analyses revealed that BBWV2 isolates could be divided largely into two groups as Group I and Group II. Based on the partial sequence analyses, 153 selected BBWV2 isolates were subgrouped into GS-I (21.6%), GS-II (3.9%) and GS-III (56.9%). BBWV2 GS-III, which was predominant in Korea, appears to be a new combination between Group I RNA-1 and Group II RNA-2. Viral disease incidence of BBWV2 on red pepper was under 2% before 2004. However, the incidence was increased abruptly to 41.3% in 2005, 58.2% in 2006 and 79% in 2007. These rapid increases might be related with the emergence of new combinations between BBWV2 groups.

Biological and molecular characterization of Soybean yellow common mosaic virus, a new species in the genus Sobemovirus

A novel soybean-infecting sobemovirus termed Soybean yellow common mosaic virus (SYCMV) was characterized. The virus has a single, positive-strand RNA genome of 4152 nucleotides. The virus contains four putative open reading frames encoding P1 (78-566 nt), polyprotein ORF2a (524-2248 nt), polymerase domain ORF2b (1852-3417 nt), and CP (3227-4030 nt). The entire nucleotide sequence of SYCMV showed 31.2-71.3% nucleotide identity with the previously known eleven species of sobemovirus. In host range analysis of SYCMV, in which twenty one species and three different Nicotiana tabacum cultivars belonging to seven families were inoculated with the virus, SYCMV had a narrow host range, infecting only Glycine max and G. soja. Based on the obtained sequence, full-length clones of SYCMV were constructed. Symptoms produced by inoculation with clones were indistinguishable from those produced by inoculation with sap from symptomatic plants. Viral RNA accumulation of SYCMV was detected in the upper leaves by Northern blotting. This indicated that full-length clones of SYCMV were sufficient to produce disease symptoms. Genomic organization, the predicted amino acid sequence, and phylogenetic analyses with known sobemoviruses confirmed the assignment of SYCMV as a new member of the genus Sobemovirus.

Development of the Large-Scale Oligonucleotide Chip for the Diagnosis of Plant Viruses and its Practical Use

A large-scale oligonucleotide (LSON) chip was developed for the detection of the plant viruses with known genetic information. The LSON chip contains two sets of 3,978 probes for 538 species of targets including plant viruses, satellite RNAs and viroids. A hundred forty thousand probes, consisting of isolate-, species- and genus-specific probes respectively, are designed from 20,000 of independent nucleotide sequence of plant viruses. Based on the economic importance, the amount of genome information, and the number of strains and/or isolates, one to fifty-one probes for each target virus are selected and spotted on the chip. The standard and field samples for the analysis of the LSON chip have been prepared and tested by RT-PCR. The probe’s specific and/or nonspecific reaction patterns by LSON chip allow us to diagnose the unidentified viruses. Thus, the LSON chip in this study could be highly useful for the detection of unexpected plant viruses, the monitoring of emerging viruses and the fluctuation of the population of major viruses in each plant.

Virus Disease Incidences and Transmission Ecology of Oriental Melons in Seongju Area

Throughout the years 2008 to 2010, we analyzed approximately two thousand oriental melon samples collected from Seongju, using electron microscopy and testing by RT-PCR using primers specific for eight cucurbit-infecting viruses. Data from RT-PCR indicated that Cucumber green mottle mosaic virus (CGMMV), Watermelon mosaic virus 2 (WMV2) and Zucchini yellow mosaic virus (ZYMV) were present and the other viruses were not detected. Among them, CGMMV and WMV2 were the most prevalent pathogens. CGMMV was thought to infect oriental melon from the early growing season, and reached nearly 100% in the later of growing period. Otherwise, WMV2 emerged from June, several months later compared to CGMMV. CGMMV was detected from all aerial parts of the oriental melon including seeds, but not from the roots of the grafted pumpkin rootstock. Seed of two out of five commercial varieties were shown to be CGMMV positive. Nine varieties of pumpkins used as rootstocks were not infected with CGMMV. When the seedlings of grafted oriental melon were transplanted into pots mixed with the oriental melon debris infected with CGMMV, they were not infected by CGMMV. Cutting of pruning shear and the contact of tendrils contributed 48% and 30% to the transmission of the virus, respectively.

Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

Identification of a New Potyvirus, Keunjorong mosaic virus in Cynanchum wilfordii and C. auriculatum

Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea(Received on October 5, 2010; Accepted on October 27, 2010)In 2006 fall, a preliminary survey of viruses in two important medicinal plants, Cynanchum wilfordii and C.auriculatum, was conducted on the experimental fields at the Agricultural Research and Extension Services ofChungbuk province in Korea. On each experimental fields, percentage of virus infection was ranged from 20to 80%, and especially an average of disease incidence propagated by roots was twice higher than that byseeds. The various symptoms were observed in Cynanchum spp. plants, such as mosaic, mottle, necrosis,yellowing, chlorotic spot and malformation etc. In electron microscopic examination of crude sap extracts,filamentous rod particles with 390-730 nm were observed in most samples. The virus particles were purifiedfrom the leaves of C. wilfordii with typical mosaic symptom, and the viral RNA was extracted from thissample containing 430-845 nm long filamentous rod. To identify the viruses, reverse transcription followed byPCR with random primers was carried out. The putative sequences of P3 and coat protein of potyvirus wereobtained. From a BLAST of the two sequences, they showed 26-38% and 62-72% identities to potyviruses,respectively. In SDS-PAGE analysis, the subunit of coat protein was approximately 30.3 kDa, close to the coatprotein of potyvirus. In bioassay with 21 species in 7 families, Chenopodium quinoa showed local lesion oninoculated leave and chlorotic spot on upper leave, but the others were not infected. RT-PCR detection usingspecific primer of C. wilfordii and C. auriculatum samples, all of 24 samples with virus symptom was positive,and five out of seven samples without virus symptom were also positive. On the basis of these data, the viruscould be considered as a new member of potyvirus. We suggested that the name of the virus was Keunjorongmosaic virus (KjMV) after the common Korean name of C. wilfordii.Keywords : Cynanchum wilfordii, Cynanchum auriculatum, Keunjorong mosaic virus, Medicinal plants,Virus disease

Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.