• The Plant Pathology Journal
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• v.17 no.6
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• pp.315-324
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• 2001

Tobacco (Nicotiana tabacum cvs.Xanthi-nc and NC 82) plants infected with Tobacco mosaic virus (TMV) were examined ultrastructurally. Local lesions produced by TMV were sunken and withered. The plants were subjected to temperature shift (TS), a method to produce programmed cell death (PCD), by placing the infected plants initially at high temperature (35$^{\circ}C$) for 2 days and then shifting them to greenhouse temperature (22-27$^{\circ}C$). As a result, expanded lesions around the original necrotic lesions were produced. The expanded area initially had no symptoms, but it withered and became necrotic 15 h after TS. No ultrastructural changes related to PCD were noted at 0 h after TS in Xanthi-nc tobacco tissues as well as in healthy and susceptible tobacco tissues infected with TMV, At 6 h after TS, chloroplasts were convoluted and cytoplasm began to be depleted; however no necrotic cells were found. At 17 h after TS, ground cytoplasm of affected cells was completely depleted and chloroplasts were stacked together with bent cell wall or dispersed in the intracellular space. Necrotic cells were also observed, containing virus particles in the necrotic cytoplasm. There were initially two types of symptoms in the expanded lesions: chlorosis and non-chlorosis (green). Abundant TMV particles and X-bodies were only found in the chlorotic tissue areas. These results suggest that PCD by TMV infection may start with the wilting of cells and tissues before necrotic lesion formation.

• The Plant Pathology Journal
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• v.18 no.2
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• pp.85-92
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• 2002

A new isolate of Cucumber mosaic virus (CMV), identified as Li-CMV was isolated from a diseased Korean native lily (Lithium tsingtauense Gilg). Biological and serological properties of Li-CMV were characterized, and reverse transcription-polymerase chain reaction (RT-PCR) analysis, restriction enzyme profiling of RT-PCR products, and nucleotide sequence analysis of RNA3 of the virus were performed in this study. Remarkable differences in symptoms between Li-CMV and ordinary CMV strains were found in tobacco plants and Datura stramonium. Li-CMV-infected tobacco plants (cv. Xanthi-nc and cv. Samsun) induced chlorotic ringspots on uninoculated upper leaves, and the symptom expression was delayed or faint whereas, ordinary CMV strains induced green mosaic symptoms on the plant. Systemic infections were observed on Nicotiana benthamiana with severe mosaic symptom. Restriction mapping analysis of RT-PCR products using MspI showed that Li-CMV belonged to CMV subgroup I. A full-length CDNA copy of RNA3 for the virus was amplified by RT-PCR, cloned, and its complete nucleotide sequence was determined. The RNA3 of Li-CMV was 2, 232 nucleotides long, and consisted of two open reading frames of 843 and 657 bases encoding 3a protein (movement protein) and coat protein, respectively. Results of this study indicate that Li-CMV is a novel strain and belongs to subgroup I of CMV in the genus Cucumovirus.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.310-314
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• 2011

Nonstructural protein 3 (NS3) encoded by RNA3 of Rice stripe virus (RSV), known to be a suppressor of gene silencing, was cloned and sequenced. The cloned NS3 gene is composed of 636 nucleotides encoding 211 deduced amino acids, and showed a high degree of similarity with the equivalent genes isolated from Korea, Japan and China. The NS3 gene promoted the enhancement of transient gene expression and suppressed transgene co-silencing. In the transient GFP expression via agroinfiltration, GFP expression was dramatically enhanced in terms of both protein yield and expression period in the presence of NS3. The highest accumulation of GFP protein reached to 6.8% of total soluble proteins, which corresponded to a two-fold increase compared to that obtained in the absence of NS3. In addition, NS3 significantly suppressed the initiation of GFP co-silencing induced by the additive GFP infiltration in GFP-transgenic Nicotiana benthamiana. The NS3 gene was also found to be a stronger suppressor than Cucumber mosaic virus 2b. These observations are believed to be derived from the strong suppressive effect of NS3 on gene silencing, and indicate that NS3 could be used as an effective enhancer for the rapid production of foreign proteins in plants.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.372-377
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• 2011

A virus causing mottle and stunt symptom on Zea mays was observed around Ulleng-do, Korea and identified as Cucumber mosaic virus (CMV-ZM) based upon biological, serological, and molecular characteristics. In host range studies, the CMV-ZM isolate produced local lesions on Datura stramonium, Vigna unguiculata, Cucurbita moschata, Chenopodium amaranticolor, Ch. quinoa, whereas this isolate produced systemic mosaic on Nicotiana tabacum cv. 'Xanthi-nc', Capsicum annuum, Solanum lycopersicum, Solanum melongena, Cucurbita pepo, and Z. mays. In addition, chlorotic local rings on inoculated leaves along with severe mosaic, malformation, and fern leaf symptoms on upper systemic leaves were shown in N. glutinosa plants. Complete nucleotide sequences of each genomic RNA segment was determined and compared to those of the other CMV strains. Comparison of the nucleotide sequence of 1a open reading frame (ORF) revealed approximately 89.2-92.4% sequence identity with each CMV subgroup IA and IB strain, while showing only 78% sequence identity with CMV subgroup II. Nucleotide sequence analysis of RNA2 ORFs revealed 85.3-97.6% sequence identity with subgroup I. In ORFs of RNA3, levels of nucleotide sequence identities were higher than 92-99.2% with CMV subgroup I and lower than 82% with CMV isolates of subgroup II. These results suggest that CMV-ZM isolate is more closely related to subgroup I than subgroup II and therefore, CMV-ZM isolate might be classified into as CMV subgroup I based on biological and molecular analysis.

• Research in Plant Disease
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• v.22 no.2
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• pp.65-71
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• 2016

Cymbidium mosaic virus (CymMV) is a major virus infecting orchid plants and causing economic loss. In this study, the incidence of viral infection in Calanthe spp. at the Korean Institute of Calanthe was investigated using reverse transcription polymerase chain reaction. The CymMV infection rate was 42%, and the two viruses Odontoglossum ringspot virus and Cucumber mosaic virus had frequencies of 8% and 2%, respectively. Additionally, we characterized an isolate of CymMV, CymMV-GW, using biological tests and examined the nucleotide sequence properties of its complete genome. CymMV-GW induced chlorotic ringspots and chlorotic spot symptoms in inoculated leaves of Chenopodium amaranticolor and Nicotiana benthamiana, respectively. In this study, we have for the first complete genome sequence of CymMV-GW in Korea. The CymMV-GW genome was 6,225 nucleotides in length, excluding the poly-(A) tail, and showed whole-genome nucleotide and amino acid sequence identities of 97.7% and 100%, respectively, with the NJ-1 isolate of CymMV. Here, we report the complete genome sequence of the CymMV-GW isolate and viral infection rates for Calanthe spp. in Korea.

• The Plant Pathology Journal
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• v.26 no.2
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• pp.139-148
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• 2010

A virus causing stunt, yellowing, severe mosaic, malformation symptoms on leaves and uneven development and malformation on fruits of zucchini was prevalent around Goseong, Gyeongsangnam-do, Korea. A survey conducted (2004) in the Goseong area revealed about 20% virus infection rate. The disease causative identified as Cucumber mosaic virus (CMV-Z1) was further characterized. The isolate induces mosaic symptoms on Cucumis sativus, while severe mosaic, stunt and malformation on C. pepo. Thin section analyses have shown that virus inclusions are formed in the cuticle layers as well as epidermal, parenchyma and collenchymas cells in virus-infected Nicotiana tabacum. CMV-Z1 isolate induced specific cytoplasmic inclusion bodies such as irregular clumps (IC), crystal (Cr) and irregular chloroplasts (ICh). IC was made up of virus particles interspersed with a darkly stained amorphous material and found both in the cytoplasm and vacuoles, whereas ICh and Cr were rarely found in the vacuoles. The genome of CMV-Z1 RNA-1 consists of 3359 nucleotide (nt) encoding 1a protein of 993 amino acids (aa). The CMV-Z1 RNA-2 was 3050 nt in length containing 2a (857 aa) and 2b (110 aa), while RNA-3 encoding 3a movement protein (279 aa) and coat protein (218 aa) was 2215 nt in length. Phylogenetic analyses of nucleotide sequences of CMV-Z1 isolate appeared it is more closely related to subgroup IA than to subgroup IB or II.

• The Plant Pathology Journal
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• v.33 no.2
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• pp.163-169
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• 2017

$SIMAPKKK{\alpha}$, a tomato (Solanum lycopersicum) mitogen-activated protein kinase kinase kinase, is a positive regulator of Pto-mediated effector-triggered immunity, which elicits programmed cell death (PCD) in plants. In this study, we examined whether putative phosphorylation sites in the conserved activation segment of the $SIMAPKKK{\alpha}$ kinase domain are critical for eliciting PCD. Three amino acids, $threonine^{353}$, $serine^{360}$ ($Ser^{360}$), or $serine^{364}$ ($Ser^{364}$), in the conserved activation segment of $SIMAPKKK{\alpha}$ kinase domain were substituted to alanine (T353A, S360A, or S364A), and these variants were transiently expressed in tomato and Nicotiana benthamiana plants. Two alanine substitutions, S360A and S364A, completely abolished $SIMAPKKK{\alpha}$ PCD-eliciting activity in both plants, while T353A substitution did not affect its PCD-eliciting activity. $SIMAPKKK{\alpha}$ wild type and variant proteins accumulated to similar levels in plant leaves. However, $SIMAPKKK{\alpha}$ protein with the largest size was missed when either S360A or S364A substitutions were expressed, whereas proteins with the smaller masses were more accumulated than those of full-length of $SIMAPKKK{\alpha}$ and T353A. These results suggest that phosphorylation of $SIMAPKKK{\alpha}$ at $Ser^{360}$ and $Ser^{364}$ is critical for PCD elicitation in plants.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.79-82
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• 2000

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which was designated NtCDPK1. Accumulation of the NtCDPK1 mRNA was stimulated by various stimuli, including phytohormones, CaCl$_2$ wounding, fungal elicitors, chitin and methyl jasmonate. The NtCDPK1 gene encodes a functional Ser/Thr protein kinase of which phosphorylation activity is strongly induced by calcium. By analyzing expression of the NtCDPK1-GFP fusion protein and by immunoblotting with antibody which reacts with NtCDPK1, we found that NtCDPK1 is localized in membrane and nucleus in plant cells. Silencing expression of the NtCDPK1 transgene resulted in marked decrease of lateral root development in the transgenic tobacco plants. Yeast two hybrid screening using NtCDPK1 as a bait identified a tobacco homologue of proteasome regulatory subunit 21D7, designated Nt21D7. The 21D7 mRNA has been shown to be predominantly expressed in proliferating tissues in the cell cycledependent manner in carrot. The recombinant NtCDPK1 protein associated with Nt21D7 in vitro, and could phosphorylate the Nt21D7 protein in vitro in the presence of calcium, suggesting that Nt21D7 protein is a natural substrate of NtCDPK1 in tobacco. These results suggest that NtCDPK1 may regulate tell proliferation processes, such as lateral root formation, by regulating specificity and/or activity of proteasome-mediated protein degradation pathway.