Jae Sun Moon

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.

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.

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.

Genomic detection and characterization of a Korean isolate of Little cherry virus 1 sampled from a peach tree

A peach tree (Prunus persica) showing yellowing and mild mottle symptoms was analyzed using high-throughput RNA sequencing to determine the causal agent. A total of nine contigs similar to Little cherry virus 1 (LChV-1) were produced, and all the contigs showed nucleotide sequence identity (lower than 83xa0%) and query coverage (higher than 73xa0%) with LChV-1. The symptomatic peach sample was confirmed to be infected with LChV-1-like virus as a result of reverse transcription-polymerase chain reaction using primers designed based on sequences of the contigs. Occurrence of diseases caused by LChV-1 in Prunus species has been reported. Complete 16,931-nt genome of the peach virus composed of eight open reading frames was determined, and conserved domains including viral methyltransferase, viral helicase 1, RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (HSP70h), HSP90h and closterovirus coat protein (CP) were identified. Phylogenetic trees based on amino acid sequence alignments between the peach virus and members in the family Closteroviridae showed that the virus was most similar to LChV-1. Pairwise comparisons based on amino acid sequence alignments of three genes (RdRp, HSP70h and CP) between the peach virus and LChV-1 isolates showed the highest amino acid sequence identities, with 84.32xa0% for RdRp, 85.48xa0% for HSP70h and 80.45xa0% for CP. These results indicate that this is the first report for the presence of LChV-1 in South Korea and may be one of the first reports of natural infection of peach by LChV-1. Although it is not clear if LChV-1 YD isolate was responsible for specific symptoms observed, detection and characterization of the peach tree-infecting LChV-1 in South Korea would be useful in terms of the epidemiology of LChV-1.

Complete genome sequence of a tentative new caulimovirus from the medicinal plant Atractylodes macrocephala

A total of nine contigs related to caulimovirus-like sequences were detected using high-throughput paired-end RNA sequencing. An attempt to find the plant sample infected with this type of virus identified the medicinal plant Atractylodes macrocephala Koidzumi showing mild mottle symptoms. Subsequently, the complete DNA genome sequence of the Atractylodes virus was determined. The 8,105-nt genome of the virus was composed of six open reading frames and displayed the highest nucleotide sequence identity (70xa0%) with soybean Putnam virus. Based upon the symptoms observed on the source plant, we propose to refer to this new member of the genus Caulimovirus as atractylodes mild mottle virus.

Complete Genome Sequences of Grapevine Yellow Speckle Viroid 1 and Hop Stunt Viroid Assembled from the Transcriptome of Ixeridium dentatum Plants.

ABSTRACT Here, we report complete genome sequences of grapevine yellow speckle viroid 1 (GYSVd1) and hop stunt viroid (HSVd), members of the family Pospiviroidae, assembled from the transcriptome data generated from Ixeridium dentatum plants. To our knowledge, this is the first report of GYSVd1 and HSVd in I. dentatum.

First Report of Peanut mottle virus Infecting Soybean in South Korea

In July 2013, soybean (Glycine max) plants at the research field in Daegu, South Korea, showed virus-like symptoms, such as mosaic, mottle, yellowing, and stunting. Overall, there were approximately 1% of soybean plants that showed these symptoms. Sixteen soybean samples were collected based on visual symptoms and subjected to laboratory characterization. Total RNA was extracted from each sample with the Tri Reagent (Molecular Research Center, Cincinnati, OH) and cDNA was synthesized using random N25 primer with RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA), according to the manufacturers instructions. All samples were tested by PCR with Prime Taq Premix (2X) (GeNet Bio, Daejeon, Korea) and primer sets specific to Soybean mosaic virus (SMV; 5-CATATCAGTTTGTTGGGCA-3 and 5-TGCCTATACCCTCAACAT-3), Peanut stunt virus (PSV; 5-TGACCGCGTGCCAGTAGGAT-3 and 5-AGGTDGCTTTCTWTTGRATTTA-3), Soybean yellow mottle mosaic virus (SYMMV; 5-CAACCCTCAGCCACATTCAACTAT-3 and 5-TCTAACCACCCCACCCGAAGGATT-3), and Soybean yellow common mosaic virus (SYCMV; 5-TTGGCTGAGAGGAGTGGCTT-3 and 5-TGCGGTCGTGTAGTCAGTG-3). Among 16 samples tested, five were positive for SMV and two for SYMMV. Three samples were found infected by both SMV and SYMMV and four by both SMV and PSV. Since two of the symptomatic samples were not infected by viruses described above, a pair of primers specific to Peanut mottle virus (PeMoV; 5-GCTGTGAATTGTTGTTGAGAA-3 and 5-ACAATGATGAAGTTCGTTAC-3) was tested (1). All 16 samples were subjected to RT-PCR with primers specific to PeMoV. Four were found positive for PeMoV. Two of them were already found infected with SYMMV. In order to identify the complete nucleotide sequences of PeMoV coat protein (CP), another primer set (5-TGAGCAGGAAAGAATTGTTTC-3 and 5-GGAAGCGATATACACACCAAC-3) was used. RT-PCR product was cloned into RBC TA Cloning Vector (RBC Bioscience, Taipei, Taiwan) and the nucleotide sequence of the insert was determined by Macrogen (Seoul, Korea). CP gene of the PeMoV (GenBank Accession No. KJ664838) showed the highest nucleotide sequence identity with PeMoV isolate Habin (KF977830; 99% identity), and the highest amino acid identity with GenBank Accession No. ABI97347 (100% identity). In order to fulfill Kochs postulates, several G. max cv. Williams 82 were inoculated with the extracts of PeMoV-infected leaf tissue. At 14 days post-inoculation, plants showed systemic mottle symptoms. These symptomatic plants were subjected to RT-PCR, and the nucleotide sequences of the PCR product were found identical to that of the virus in the inoculum. To our knowledge, this is the first report of soybean-infecting PeMoV, a member of the genus Potyvirus in the family Potyviridae, in South Korea. Reference: (1) R. G. Dietzgen et al. Plant Dis. 85:989, 2001.

First Report of Soybean yellow mottle mosaic virus in Soybean in North America

Soybean yellow mottle mosaic virus (SYMMV) is a soybean-infecting virus recently discovered in Korea that initially induces bright yellow mosaic on leaves followed by stunting and reduced growth of older leaves (1). Nucleotide sequence analysis of genomic RNA of the Korean SYMMV isolate suggested that the virus is a new member of the genus Carmovirus in the family Tombusviridae. To determine whether SYMMV is present in the United States, single leaflets were collected without regard for symptoms from 7 to 10 plants in each of 136 plots in August 2008 from a research field in Stoneville, MS that contained 16 plant introductions (including five from Korea) and Williams 82. Samples were grouped into 10 pools of 100 leaves from which total RNA was extracted with the Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScript III Platinum SYBR Green One-Step Quantitative Real-time Reverse Transcriptase-PCR Kit (Invitrogen, Carlsbad, CA) and two pairs of oligonucleotide primers (5-CGTCTGCCAGGGTTTAATACTA-3, and 5-GATTAGCATGTCAGGGTGGTCG-3; and 5-ACTGAGTCCCCTGCTTAT-3 and 5-CATCACTAGCGTCYGGATCA-3) that were designed from regions conserved between SYMMV and Cowpea mottle virus (CPMoV; a related and seed-transmitted carmovirus). Six 100-leaflet pools were positive with both primer sets and four pools were negative with both primer sets. Total RNA extracted from one positive pool was reverse transcribed using SuperScript II reverse transcriptase and a primer complementary to nt 4,000 to 4,009 of the SYMMV genome and amplified using iProof DNA polymerase (Bio-Rad, Hercules, CA) as two overlapping DNA fragments using primers corresponding to nt 1 to 21 and complementary to nt 3,483 to 3,508 and corresponding to nt 3,366 to 3,391 and complementary to nt 4,000 to 4,009. DNA fragments were sequenced using a BigDye Terminator Cycle Sequencing Kit and ABI 3730XL capillary sequencers (Applied Biosystems, Foster City, CA). The 4,009-nt sequence of the Mississippi SYMMV isolate (GenBank Accession No. FJ707484) was 96% identical to the Korean SYMMV isolate and 65% identical to CPMoV. Because of the sampling techniques used, it was not possible to associate SYMMV-positive plants with disease symptoms in Mississippi. To our knowledge, this is the first report of SYMMV in North America. Reference: (1) M. Nam et al. Online publication. doi:10.1077/s00705-009-0480. Arch. Virol., 2009.

Nucleotide sequence and genome organization of a new proposed crinivirus, tetterwort vein chlorosis virus

The genome of tetterwort vein chlorosis virus (TVCV) from South Korea has been completely sequenced. Its genomic organization resembles those of other criniviruses, with several new features, indicating that TVCV is a member of a new species in the genus Crinivirus, family Closteroviridae. RNA1 contains 8467 nucleotides, with at least four opening reading frames (ORFs). ORF1a encodes a protein with predicted papain-like protease, methyltransferase, and helicase activities. ORF1b encodes a putative RNA-dependent RNA polymerase that is apparently expressed through a +1 ribosomal frameshift. RNA2 contains 8113 nucleotides encoding at least nine proteins, similar to most crinivirus RNA2s. The 3′ untranslated regions of the bipartite RNA genome share 82.1xa0% nucleotide sequence identity.

Complete genome sequence of keunjorong mosaic virus, a potyvirus from Cynanchum wilfordii

We have determined the complete genome sequence of keunjorong mosaic virus (KjMV). The KjMV genome is composed of 9,611 nucleotides, excluding the 3′-terminal poly(A) tail. It contains two open reading frames (ORFs), with the large one encoding a polyprotein of 3,070 amino acids and the small overlapping ORF encoding a PIPO protein of 81 amino acids. The KjMV genome shared the highest nucleotide sequence identity (57.5 xa0%) with pepper mottle virus and freesia mosaic virus, two members of the genus Potyvirus. Based on the phylogenetic relatedness to known potyviruses, KjMV appears to be a member of a new species in the genus Potyvirus.