Characterization of the in vitro Activities of the P1 and Helper Component Proteases of Soybean mosaic virus Strain G2 and Tobacco vein mottling virus

Lim, Hyoun-Sub,Jang, Chan-Yong,Nam, Ji-Ryun,Li, Meijia,Hong, Jin-Sung,Bae, Han-Hong,Ju, Ho-Jong,Kim, Hong-Gi,Ford, Richard E.,Domier, Leslie L. The Korean Society of Plant Pathology 2012 Plant Pathology Journal Vol.28 No.2

Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these proteases. The in vitro activities of two of the proteases of Soybean mosaic virus (SMV) and Tobacco vein mottling virus (TVMV) were compared in the rabbit reticulocyte lysate in vitro translation system using synthetic RNA transcripts. Transcripts produced from SMV and TVMV cDNAs that included the P1 and helper component-protease (HC-Pro) coding regions directed synthesis of protein products that were only partially processed. Unprocessed poly-proteins were not detected from transcripts that included all of the P1, HC-Pro, P3 and portions of the cylindrical inclusion protein coding regions of either virus. Addition of soybean trypsin inhibitor to in vitro translation reactions increased the accumulation of the unprocessed polyprotein from TVMV transcripts, but did not alter the patterns of proteins produced from SMV. These experiments suggest that SMV-and TVMV-encoded proteases are differentially sensitive to protease inhibitors.

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

Lim, Hyoun-Sub,Lee, Mi Yeon,Moon, Jae Sun,Moon, Jung-Kyung,Yu, Yong-Man,Cho, In Sook,Bae, Hanhong,DeBoer, Matt,Ju, Hojong,Hammond, John,Jackson, Andrew O. The Korean Society of Plant Pathology 2013 Plant Pathology Journal Vol.29 No.1

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) treat-ments 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.

Soybean mosaic virus Infection and Helper Component-protease Enhance Accumulation of Bean pod mottle virus-Specific siRNAs

Lim, Hyoun-Sub,Jang, Chan-Yong,Bae, Han-Hong,Kim, Joon-Ki,Lee, Cheol-Ho,Hong, Jin-Sung,Ju, Ho-Jong,Kim, Hong-Gi,Domier, Leslie L. The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.4

Soybean plants infected with Bean pod mottle virus (BPMV) develop acute symptoms that usually decrease in severity over time. In other plant-virus interactions, this type of symptom recovery has been associated with degradation of viral RNAs by RNA silencing, which is accompanied by the accumulation of virus-derived small interfering RNAs (siRNAs). In this study, changes in the accumulation of BPMV siRNAs were investigated in soybean plants infected with BPMV alone, or infected with both BPMV and Soybean mosaic virus (SMV) and in transgenic soybean plants expressing SMV helper component-protease (HC-Pro). In many potyviruses, HC-Pro is a potent suppressor of RNA silencing. In plants infected with BPMV alone, accumulation of siRNAs was positively correlated with symptom severity and accumulation of BPMV genomic RNAs. Plants infected with both BPMV and SMV and BPMV-infected transgenic soybean plants expressing SMV HC-Pro exhibited severe symptoms characteristic of BPMVSMV synergism, and showed enhanced accumulation of BPMV RNAs and siRNAs compared to plants infected with BPMV alone and nontransgenic plants. Likewise, SMV HC-Pro enhanced the accumulation of siRNAs produced from a silenced green fluorescent protein gene in transient expression assays, while the P19 silencing suppressor of Tomato bushy stunt virus did not. Consistent with the modes of action of HC-Pro in other systems, which have shown that HC-Pro suppresses RNA silencing by preventing the unwinding of duplex siRNAs and inhibiting siRNA methylation, these studies showed that SMV HC-Pro interfered with the activities of RNA-induced silencing complexes, but not the activities of Dicer-like enzymes in antiviral defenses.

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Lim, Hyoun-Sub,Bragg, Jennifer N.,Ganesan, Uma,Ruzin, Steven,Schichnes, Denise,Lee, Mi Yeon,Vaira, Anna Maria,Ryu, Ki Hyun,Hammond, John,Jackson, Andrew O. American Society for Microbiology 2009 Journal of virology Vol.83 No.18

<B>ABSTRACT</B><P><I>Barley stripe mosaic virus</I> (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used <I>Agrobacterium tumefaciens</I>-mediated protein expression in <I>Nicotiana benthamiana</I> leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.</P>

Characteristics of a Lettuce mosaic virus Isolate Infecting Lettuce in Korea

Lim, Seungmo,Zhao, Fumei,Yoo, Ran Hee,Igori, Davaajargal,Lee, Su-Heon,Lim, Hyoun-Sub,Moon, Jae Sun The Korean Society of Plant Pathology 2014 Plant Pathology Journal Vol.30 No.2

Lettuce mosaic virus (LMV) causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMV-RoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup) than to LMV-E (LMV-RoW group but not LMV-Common subgroup) even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

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

Lim, Seungmo,Igori, Davaajargal,Yoo, Ran Hee,Zhao, Fumei,Cho, In-Sook,Choi, Gug-Seoun,Lim, Hyoun-Sub,Lee, Su-Heon,Moon, Jae Sun M. Nijhoff ; Kluwer Academic Publishers 2015 Virus genes Vol.51 No.2

<P>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 83??%) and query coverage (higher than 73??%) 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 84.32??% for RdRp, 85.48??% for HSP70h and 80.45??% 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.</P>

Fungal Endophytes from Three Cultivars of Panax ginseng Meyer Cultivated in Korea

Sang Un Park,Hyoun-Sub Lim,Kee-Choon Park,Young-Hwan Park,Hanhong Bae 고려인삼학회 2012 Journal of Ginseng Research Vol.36 No.1

In order to investigate the diversity of endophytes, fungal endophytes in Panax ginseng Meyer cultivated in Korea were isolated and identifi ed using internal transcribed spacer (ITS) sequences of ribosomal DNA. Three cultivars of 3-year-old ginseng roots (Chunpoong, Yunpoong, and Gumpoong) were used to isolate fungal endophytes. Surface sterilized ginseng roots were placed on potato dextrose agar plates supplemented with ampicilin and streptomycin to inhibit bacterial growth. Overall, 38 fungal endophytes were isolated from 12 ginseng roots. According to the sequence analysis of the ITS1-5.8S-ITS2, 38 fungal isolates were classifi ed into 4 different fungal species, which were Phoma radicina, Fusarium oxysporum, Setophoma terrestris and Ascomycota sp. 2-RNK. The most dominant fungal endophyte was P. radicina in 3 cultivars. The percentage of dominant endophytes of P. radicina was 65.8%. The percentage of colonization frequency of P. radicina was 80%, 52.9%, and 75% in Chunpoong, Yunpoong, and Gumpoong, respectively. The second most dominant fungal endophyte was F. oxysporum. The diversity of the fungal endophytes was low and no ginseng cultivar specifi city among endophytes was detected in this study. The identifi ed endophytes can be potential fungi for the production of bioactive compounds and control against ginseng pathogens.

Fungal Endophytes from Three Cultivars of Panax ginseng Meyer Cultivated in Korea

Park, Sang-Un,Lim, Hyoun-Sub,Park, Kee-Choon,Park, Young-Hwan,Bae, Han-Hong The Korean Society of Ginseng 2012 Journal of Ginseng Research Vol.36 No.1

In order to investigate the diversity of endophytes, fungal endophytes in Panax ginseng Meyer cultivated in Korea were isolated and identified using internal transcribed spacer (ITS) sequences of ribosomal DNA. Three cultivars of 3-year-old ginseng roots (Chunpoong, Yunpoong, and Gumpoong) were used to isolate fungal endophytes. Surface sterilized ginseng roots were placed on potato dextrose agar plates supplemented with ampicilin and streptomycin to inhibit bacterial growth. Overall, 38 fungal endophytes were isolated from 12 ginseng roots. According to the sequence analysis of the ITS1-5.8S-ITS2, 38 fungal isolates were classified into 4 different fungal species, which were Phoma radicina, Fusarium oxysporum, Setophoma terrestris and Ascomycota sp. 2-RNK. The most dominant fungal endophyte was P. radicina in 3 cultivars. The percentage of dominant endophytes of P. radicina was 65.8%. The percentage of colonization frequency of P. radicina was 80%, 52.9%, and 75% in Chunpoong, Yunpoong, and Gumpoong, respectively. The second most dominant fungal endophyte was F. oxysporum. The diversity of the fungal endophytes was low and no ginseng cultivar specificity among endophytes was detected in this study. The identified endophytes can be potential fungi for the production of bioactive compounds and control against ginseng pathogens.

Determination of Intramuscular Fat Content in Beef using Magnetic Resonance Imaging

Lee, Sangdae,Lohumi, Santosh,Lim, Hyoun-Sub,Gotoh, Takafumi,Cho, Byoung-Kwan,Jung, Samooel FACULTY OF AGRIC PUBLICATIONS-KYUSHU UNIV 2015 Journal of the Faculty of Agriculture, Kyushu Univ Vol. No.

Update on the Effects of Sound Wave on Plants

Chowdhury, Md. Emran Khan,Lim, Hyoun-Sub,Bae, Hanhong The Korean Society of Plant Pathology 2014 식물병연구 Vol.20 No.1

Plant growth is considered the sum of cell proliferation and subsequent elongation of the cells. The continuous proliferation and elongation of plant cells are vital to the production of new organs, which have a significant impact on overall plant growth. Accordingly, the relationship between environmental stimuli, such as temperature, light, wind, and sound waves to plant growth is of great interest in studies of plant development. Sound waves can have negative or positive effects on plant growth. In this review paper we have summarized the relationship between sound waves and plant growth response. Sound waves with specific frequencies and intensities can have positive effects on various plant biological indices including seed germination, root elongation, plant height, callus growth, cell cycling, signaling transduction systems, enzymatic and hormonal activities, and gene expression.