In Sook Cho

Insights into Alternanthera mosaic virus TGB3 Functions: Interactions with Nicotiana benthamiana PsbO Correlate with Chloroplast Vesiculation and Veinal Necrosis Caused by TGB3 Over-Expression

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.

Tyrosinase inhibitory components from Aloe vera and their antiviral activity

Abstract A new compound, 9-dihydroxyl-2-O-(Z)-cinnamoyl-7-methoxy-aloesin (1), and eight known compounds (2–9) were isolated from Aloe vera. Their structures were elucidated using 1D/2D nuclear magnetic resonance and mass spectra. Compound 9 exhibited reversible competitive inhibitory activity against the enzyme tyrosinase, with an IC50 value of 9.8u2009±u20090.9u2009µM. A molecular simulation revealed that compound 9 interacts via hydrogen bonding with residues His244, Thr261, and Val283 of tyrosinase. Additionally, compounds 3 and 7 were shown by half-leaf assays to exhibit inhibitory activity towards Pepper mild mottle virus.

Tomato spotted wilt virus Isolates Giving Different Infection in Commercial Capsicum annuum Cultivars

Tomato spotted wilt virus (TSWV)-infected Capsicum annuum plants were collected from open fields during June to July in 2010. The TSWV isolates were designated as Gneung, Ghang-Kjj, Gchang-Njc, Ghae, and Pap. The nucleotide sequence of the nucleocapsid protein (N) and movement protein (NSm) of the five isolates was determined. The pathogenicity of the five isolates was determined on 14 C. annuum cultivars two times by using mechanical inoculation. The five isolates induced different response: Both Gneung and Gchang-Kjj did not infect any of the cultivars in the 2nd trial, while Gchang-Njc, Ghae and Pap infected 11, 6 and 13 of 14 cultivars, respectively. The five isolates also were tested on Solanum lycopersicum breeding line TGC09-71 and three Nicotiana species. S. lycopersicum showed a similar response to the five isolates as did C. annuum. Both Gchang-Njc and Ghae infected systemically all three Nicotiana species tested. While both Pap and Gneung did not infect any of the Nicotiana species tested. In conclusion, five TSWV isolates induced different infection spectra in C. annuum cultivars, Nicotiana species and an S. lycopersicum breeding line. Amino acid sequence analysis of the NSm gene could not support or explain the different infection spectra of the five isolates. This study indicated that various isolates must be used as virus inocula for evaluation of C. annuum and S. lycopersicum cultivars in breeding programs for TSWV resistance.

First Report of Tomato spotted wilt virus in Brugmansia suaveolens in Korea

Brugmansia suaveolens, also known as angels trumpet, is a semi-woody shrub or a small tree. Because flowers of B. suaveolens are remarkably beautiful and sweetly fragrant, B. suaveolens is grown as ornamentals outdoors year-round in the tropics and subtropics, and as potted plants in temperate regions (1). In February 2013, virus-like symptoms including mosaic symptoms followed by distortion of leaves were observed in a potted B. suaveolens in a nursery in Chung-Nam Province, Korea. Symptomatic leaves were analyzed for the presence of several ornamental viruses including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato bush stunt virus (TBSV), and Tomato spotted wilt virus (TSWV) by immune-strip diagnostic kits that were developed by our laboratory. Positive controls and extract from healthy leaves of B. suaveolens as a negative control were included in each immune-strip assay. TSWV was detected serologically from the naturally infected B. suaveolens, but CMV, TBSV, and TMV were not detected from the B. suaveolens. The presence of TSWV (named TSWV-AT1) was confirmed by commercially available double-antibody sandwich (DAS)-ELISA kits (Agdia, Elkhart, IN). TSWV-AT1 was mechanically transmitted from the ELISA-positive B. suaveolens to Capsicum annuum and Nicotiana glutinosa, respectively. Inoculated C. annuum showed chlorotic rings in the inoculated leaves and inoculated N. glutinosa produced mosaic and systemic necrosis in the inoculated leaves after 7 days inoculation, respectively, which were consistent with symptoms caused by TSWV (2). To confirm further TSWV-AT1 infection, reverse transcription (RT)-PCR was performed using the One-Step RT-PCR (Invitrogen, Carlsbad, CA) with TSWV-specific primers, TSWV-NCP-For and TSWV-NCP-Rev (3), designed to amplify a 777-bp cDNA of the nucleocapsid protein (NCP) gene. Total RNAs from naturally infected B. suaveolens, symptomatic C. annuum, and N. glutinosa were extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Total RNAs obtained from a Korean isolate of TSWV (Accession No. JF730744) and healthy B. suaveolens were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic B. suaveolens, C. annuum, and N. glutinosa but not from healthy leaves of B. suaveolens. The amplified RT-PCR product from TSWV-AT1 was directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the TSWV-AT1 NCP sequence (AB910533) with NCP sequences of other TSWV isolates using MEGA5 software (4) revealed 99.0% aa identity with an Korean TSWV isolate (AEB33895) originating from tomato. These results provide additional confirmation of TSWV-AT1 infection. It is known that high-value ornamentals may act also as reservoirs for TSWV that can infect other ornamentals and cultivated crops, because TSWV has a very broad host range (2). Elaborate inspections for TSWV and other viruses are necessary for production of healthy B. suaveolens, since the popularity and economic importance of this ornamental plant is increasing. To our knowledge, this is the first report of TSWV in B. suaveolens in Korea. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) B.-N. Chung et al. Plant Pathol. J. 28:87, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

α-Glucosidase inhibition by prenylated and lavandulyl compounds from Sophora flavescens roots and in silico analysis

The enzyme α-glucosidase is a good drug target for the treatment of diabetes mellitus. Four minor flavonoids (1-4) from roots of Sophora flavescens showed the inhibitory activity, with IC50 values ranging from 11.0±0.3 to 50.6±1.3μM, toward α-glucosidase. An enzyme kinetics analysis of them revealed that the compounds 1 and 4 were non-competitive, and compounds 2 and 3 were un-competitive inhibitors. For molecular docking, 3-dimensional structure of α-glucosidase was built by homology modeling. As the result, four compounds 1-4 were confirmed to interact into common binding site of α-glucosidase. In addition, all of the four prenylated and lavandulyl compounds (1-4) were abundant in an ethyl acetate fraction separated from a methanol extract, and the potential inhibitor (3) was extracted best using tetrahydrofuran.

First report of Blueberry red ringspot virus infecting highbush blueberry in Korea.

Blueberry red ringspot virus (BRRSV) of the Soymovirus genus in the family Caulimovididae causes red ringspot diseases in highbush blueberry (Vaccinium corymbosum L.) on leaves, stems, and fruits. The virus has been identified in the United States, Japan, Czech Republic, Slovenia, and Poland (1). In July 2010, highbush blueberry with red ringspots on leaves and circular blotches on ripening fruits was found in one plant of cv. Duke in Pyeongtaek, Korea. The symptoms were similar to red ringspot disease caused by BRRSV (3), although stems did not show any characteristic symptoms. Red ringspots on the upper surface of leaves were the most visible symptom and became more prominent as leaves matured in August through October. Leaves of the symptomatic plant were collected and tested for BRRSV infection by PCR, and were also embedded for electron microscopy. DNA was extracted from leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturers instructions. Primer pairs BR1512F/BR2377R (5-ACAGGACGATTAGAAGATGG-3/5-CCTTTAGGGCAATATTTCTG-3, amplifying a fragment of the coat protein region with an expected size of 865 bp) and BR2961F/BR3726R (5-ACCGATACATCACAGTTCAC-3/5-TGGTTGTGATAAGATGATTCC-3, amplifying a fragment of the reverse transcriptase region with an expected size of 766 bp) were used to amplify the indicated region of BRRV in PCR. Primers were designed on the basis of the BRRSV isolate from New Jersey (GenBank Accession No. AF404509). DNA fragments of the expected sizes were obtained from the symptomatic plant, while no amplification products were obtained from highbush blueberry without symptoms. The PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of obtained fragments revealed 91 to 98% nucleotide sequence identity with the coat protein gene (GenBank Accession No. JQ706341) and 96 to 98% nucleotide sequence identity with the reverse transcriptase gene (GenBank Accession No. JQ706340) of known BRRV isolates. Electron microscopy of thin sections revealed particles approximately 50 nm diameter within electron-dense inclusion bodies, characteristic of BRRSV (2) To our knowledge, this is the first report of BRRSV infection of highbush blueberry in Korea. Highbush blueberries are usually propagated by cutting, so BRRSV suspicious plants should be tested with PCR before they are propagated. References: (1) E. Kalinowska et al. Virus Genes. DOI 10.1007/s11262-011-0679-4, 2011. (2) K. S. Kim et al. Phytopathology 71:673, 1981. (3) M. Isogai et al. J. Gen. Plant Pathol. 75:140, 2009.

First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea. References: (1) R. Li and R. Mock. Arch. Virol. 153:973, 2008. (2) R. Li and R. Mock. J. Virol. Methods 129:162, 2005.

Occurrence of Stone Fruit Viruses on Peach Trees (Prunus persica L. Batsch) in Korea

To investigate the occurrence of viruses in peach, leaf samples were collected from peach trees in commercial orchard of six areas in Korea. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify the presence of the following stone fruit viruses: Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and Plum pox virus (PPV). About 65.0% of the 515 samples were infected with ACLSV and PNRSV. Virus-like symptoms showing mosaic on leaves was observed in ACLSV infected peach trees. However, PNRSV infected peach trees showed no symptoms. These viral DNAs by sequence analysis were confirmed 4 ACLSV isolates and 3 PNRSV isolates. The Korean peach isolates of ACLSV and PNRSV showed 70-99% and 88-99% amino acid sequence identities, respectively, with those reported previously and their amino acid sequence identities with each other were approximately 95% and 88%, respectively. Phylogenetic analysis indicated that the Korean ACLSV isolates belong to the A group of ACLSV. The Korean PNRSV isolates reported in this study were grouped into I (PV32), II (PV96) and III (PE5) groups.

First Report of Cucumber mosaic virus in Catharanthus roseus in Korea

Catharanthus roseus, commonly known as Madagascar rosy periwinkle (also called vinca), is a tropical perennial herb of the family Apocyanaceae. Periwinkle is a bedding plant widely used in Korea because of its drought tolerance, low maintenance, and varied flower colors. In May 2013, virus-like foliar symptoms, including a mosaic with malformation of leaves, were observed on a periwinkle plant in a greenhouse located in Chonbuk Province, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with an immune-strip kit developed by our laboratory. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-Vin) caused necrotic local lesions on Chenopodium amaranticolor at 5 days-post-inoculation (dpi), while mild to severe mosaic was observed in Capsicum annuum, Cucumis sativus, Cucurbita pepo Cheonggobong, Nicotiana glutinosa, N. tabacumSamsun NN, Physalis angulate, and Solanum lycopersicum Pink-Top 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ~28 nm in diameter, respectively. To verify whether CMV was the causal agent for the disease symptoms observed in naturally infected periwinkle, virus-free periwinkle (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-Vin. At 6 weeks after inoculation, all plants produced systemic mosaic and distortion of leaves, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated periwinkle plants remained symptomless and virus-free. The presence of CMV-Vin in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CMVCPFor and CMVCPRev (1), which amplified the entire CP gene. RT-PCR products (657 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from a positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-Vin CP sequence (Accession No. AB910598) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-Vin infection. Being perennial, periwinkle plants could serve as a reservoir for CMV to infect other ornamentals and cultivated crops (2). To our knowledge, this is the first report of CMV infection on periwinkle in Korea. References: (1) S. K. Choi et al. Virus Res. 158:271, 2011. (2) P. Palukaitis et al. Adv. Virus. Res. 41:281, 1992.