Jin-Sung Hong

Molecular evidence supporting the confirmation of Maracuja mosaic virus as a species of the genus Tobamovirus and production of an infectious cDNA transcript

Summary.The complete genome sequence of maracuja mosaic virus (MarMV) was determined and analyzed. The full MarMV genome consisted of 6794 nucleotides, and this is the largest genome size among known tobamoviruses. The MarMV genome RNA contained four open reading frames (ORFs) coding for proteins of M(r) 126, 181, 34 and 18 kDa from the 5′ to 3′ end, respectively. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 54 and 177 nucleotides, respectively. Phylogenetic tree analysis revealed that these MarMV-encoded proteins are related to members of the Malvaceae- and Cucurbitaceae-infecting tobamoviruses. MarMV is different from other tobamoviruses and forms a new Passifloraceae-infecting subgroup. Western blot analysis showed that MarMV cross-reacted strongly with antibodies against Kyuri green mottle mosaic virus and Hibiscus latent Singapore virus. Synthesized capped transcripts from full-length cDNA of MarMV were infectious. These data clearly indicate that MarMV belongs to a separate species of the genus Tobamovirus.

Infection of soybean by cucumber mosaic virus as determined by viral movement protein

Summary.To characterize the host range determinant of the soybean strain of Cucumber mosaic virus (CMV) we analyzed a series of pseudorecombinants and chimeric viruses between infectious transcripts from two soybean strains (CMV-SC and CMV-SD) and an ordinary strain (CMV-Y). CMV-Y could not infect soybeans, even locally. Systemic infection of the two soybean-adapted soybean isolates on soybean plants mapped to RNA3. Chimeric RNA3s from between CMV-SC and CMV-Y, and chimeric RNA3s from between CMV-SC and CMV-SD, were made and inoculated onto wild soybean Iwate and soybean cv. Tsurunoko. The 3a region determined the viral systemic movement in the plants. In the wild soybean ecotype Hyougo, cell-to-cell movement of two different CMV soybean strains, one of which infects systemically while the other does not, in the inoculated leaves were almost the same, suggesting that the resistance of soybean operates at the level of long-distance movement. Our results clearly suggest that movement protein is a host determinant of CMV soybean strains.

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses.

Summary.The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3′ end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.

Variation in the Pathogenicity of Lily Isolates of Cucumber mosaic virus

Two isolates of Cucumber mosaic virus (CMV) originated from lily plants, named Ly2-CMV and Ly8-CMV, were compared with their pathological features in several host plants. Ly2-CMV and Ly8-CMV could induce systemic mosaic symptom in Nicotiana benthamiana, but Ly2-CMV could not systemically infect tomato and cucumber plants that have been used for CMV-propagative hosts. While Fny-CMV used as a control infected systemically the same host plants, producing typical CMV symptoms. Ly8-CMV could infect systemically two species of tobacco (N. tabacum cv. Xanthi-nc and N. glutinosa) and zucchini squash (Curcubita pepo), but Ly2 failed systemic infection on these plants. As resulted from tissue-print immunoblot assay, different kinetics of systemic movement between Ly2-CMV and Ly8-CMV were crucial for systemic infection in tobacco (cv. Xanthi-nc). Sequence analysis of full-length genome of two lily isolates showed Ly2 and Ly8 belonged to subgroup IA of CMV. The lily isolates shared overall 98 % sequence identity in their genomes. Coat protein, 3a protein, and 2b protein involved in virus movement was highly conserved in genomes of the isolates Ly2 and Ly8. Although there is the low frequency of recombinants and reassortants in natural CMV population, phylogenetic analysis of each viral protein among a number of CMV isolates suggested that genetic variation in a defined population of CMV lily isolates was stochastically produced.

Molecular Characterization and Infectious cDNA Clone of a Korean Isolate of Pepper mild mottle virus from Pepper

A Korean isolate of Pepper mild mottle virus (PMMoV-Kr) was isolated from a diseased hot pepper plant and its biological and molecular properties were compared to that of PMMoV-J and PMMo V -So The genomic RNA of PMMoV-Kr consists of 6,356 nucleotides. The nucleotide and amino acid sequences identities of four viral proteins and two noncoding regions among PMMoV-Kr, PMMoV-S and PMMoV-J were , respectively. Full-length cDNA amplicon of PMMoV-Kr was directly amplified by RT-PCR with a set of 5`-end primer anchoring T7 RNA promoter sequence and 3`-end virus-specific primer. Capped transcript RNAs from the full-length cDNA clone were highly infectious and caused characteristic symptoms of wild type PMMoV when mechanically inoculated to systemic host plants such as Nicotiana benthamiana and pepper plants.

Patterns in disease progress and the influence of single and multiple viral infections on pepper ( Capsicum annuum L.) growth

The patterns and progress of disease caused by multiple infections of Cucumber mosaic virus (CMV), Pepper mild mottle virus (PMMoV) and Pepper mottle virus (PepMoV) and their effects on growth of pepper plants (Capsicum annuum L.) were investigated in this study. Each virus induced distinct symptoms, but more severe symptoms, including reduced growth rates, were observed when pepper plants were simultaneously infected by more than one virus. When CMV was included in multiple viral inoculations, co-inoculations and sequential inoculations, PepMoV and PMMoV symptoms were observed but the symptoms characteristic of CMV were not masked, even though CMV titre did not increase greatly. In multiple viral infections, PepMoV titre and CMV did not increase significantly, but PMMoV titre gradually increased in most cases. Growth rates of pepper plants were greatly reduced during the 30 to 40-day post-inoculation period under both single-infection and multiple-infection conditions, but multiple viral infections of CMV pre-inoculated peppers were affected to a greater extent. A significant reduction in fruit size and fruit number was observed in single and multiple viral inoculations, and fruit malformation rates were high in CMV single-infection and multiple viral infections with CMV.

Complete genome sequence of an isolate of Pepper veinal mottle virus and phylogenetic relationship with other potyviruses.

Pepper veinal mottle virus (PVMV) is a virus species in the genus Potyvirus in the family Potyviridae [4]. The virions of members of this genus are flexuous filaments containing a single molecule of linear, positive-sense, single-stranded ribonucleic acid (ssRNA), about 9.7 kb in size, which has a poly (A) tract at the 30-end [1, 3, 12, 18, 20]. The potyviruses are transmitted by aphids in a non-persistent manner and are transmissible experimentally by mechanical inoculation [17]. PVMV, one of pepper-infecting potyviruses, is widespread in African countries and causes veinal yellowing and mottling symptoms in peppers [2, 9] and various weeds such as Physalis angulata [6]. PVMV was first isolated from Capsicum annuum and Capsicum frutescens from Ghana in 1971 [4], and this virus was serologically distinguished as a potyvirus distinct from potato virus Y (PVY) [4]. PVMV causes mosaic and stunt disease in C. frutescens and induces chlorotic local lesions in Chenopodium amaranticolar, Nicotiana tabacum cv. Xanthi and N. tabacum cv. Samsun [2, 4]. The symptoms of infection differ depending on the isolates and host plants [19]. Pepper-infecting potyviruses have been reported from many countries. To date, the properties and complete genome sequences of isolates of Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), Chilli veinal mottle virus (ChiVMV), PVMV and various solanaceae-infecting potyviruses (PVY, PTMV, TEV) have been reported [1, 3, 18]. Partial genome sequences of PVMV obtained from various isolates have been reported in some regions [2, 7, 8, 11], but there is no complete genome RNA sequence of PVMV so far. In this study, the full genome sequence and genome structure of a PVMV isolate were determined and compared to those of various PVMV isolates and other potyviruses. A stock culture of PVMV was obtained from the Plant Virus GenBank (Seoul, Korea) which had previously been obtained from the DSMZ (PV-0257, Germany). The isolate was originally isolated from virus-infected C. frutescens in Ghana [4]. The virus used in this study, termed PVMV-P (pepper isolate), was propagated on N. benthamiana. PVMV particles examined by electron microscopy were filamentous, about 770-nm long and 12-nm wide (data not shown), which is the typical morphology described [2]. The virus was purified from virus-infected N. benthamiana leaves by differential centrifugation [7, 12]. Viral genomic RNA was isolated using sodium dodecyl sulphate (SDS)/ proteinase K and phenol extraction method [12, 13] and was used as template for RT-PCR. The RT-PCR was performed in a reaction mixture containing 10 pM of potyvirus-specific primer targeted to the NIb and CP regions, and oligo dT primer [15, 17]. PCR was performed in a thermal cycler for 3 min at 94 C, for a total of 35 cycles (94 C for 40 s, 52 C for 1 min, 72 C for 1 min), and final extension at 72 C for 10 min. The partial sequence of PVMV was determined using the amplified PCR products (GenBank: AB126177). Nucleotide sequences at the 50-end for PVMV were determined by 50-rapid amplification of cDNA ends (RACE) using PVMV 50 RACE (50-GTGAGTGTTGTA GAAGCACGGG-30) and the SMARTII oligo nucleotides (SMART RACE cDNA amplification kit, Clontech, CA, USA). Selected clones and cDNAs were then sequenced J. H. Ha J. S. Hong K. H. Ryu (&) Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women’s University, Seoul 139-774, South Korea e-mail: ryu@swu.ac.kr

Characterization of an Isolate of Cucumber mosaic virus Isolated from Chinese aster (Callistephus chinensis)

An isolate of Cucumber mosaic virus (CMV), designated as Cas-CMV, was isolated from Chinese aster (Callistephus chinensis) showing severe mosaic symptom, and its properties was compared to the well-characterized Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, and restriction enzyme profile of the PCR products. Cas-CMV differed markedly in their host reaction to Fny-CMV or As-CMV in Cucurbita pepo cv. Black beauty. In the zucchini squash, all strains induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves. However, symptoms induced by Cas-CMV were developed lethal necrosis on the young plants 15 to 20 days after inoculation. In experiments of dsRNA analysis and RT-PCR analysis, properties of Cas-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using HindIII of the RT-PCR products showed that Cas-CMV belong to a member of CMV subgroup IA.

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca2+-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.

Phylogenetics and Gene Structure Dynamics of Polygalacturonase Genes in Aspergillus and Neurospora crassa

Polygalacturonase (PG) gene is a typical gene family present in eukaryotes. Forty-nine PGs were mined from the genomes of Neurospora crassa and five Aspergillus species. The PGs were classified into 3 clades such as clade 1 for rhamno-PGs, clade 2 for exo-PGs and clade 3 for exo- and endo-PGs, which were further grouped into 13 sub-clades based on the polypeptide sequence similarity. In gene structure analysis, a total of 124 introns were present in 44 genes and five genes lacked introns to give an average of 2.5 introns per gene. Intron phase distribution was 64.5% for phase 0, 21.8% for phase 1, and 13.7% for phase 2, respectively. The introns varied in their sequences and their lengths ranged from 20 bp to 424 bp with an average of 65.9 bp, which is approximately half the size of introns in other fungal genes. There were 29 homologous intron blocks and 26 of those were sub-clade specific. Intron losses were counted in 18 introns in which no obvious phase preference for intron loss was observed. Eighteen introns were placed at novel positions, which is considerably higher than those of plant PGs. In an evolutionary sense both intron loss and gain must have taken place for shaping the current PGs in these fungi. Together with the small intron size, low conservation of homologous intron blocks and higher number of novel introns, PGs of fungal species seem to have recently undergone highly dynamic evolution.