• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.220-228
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• 2017

Many crops including cereals, tuber crops, feeds, and turf grasses are often damaged by various environmental stresses such as drought, salt, cold, and high temperature, causing the reduction of their productivity. Plants are sessile and cannot escape from environmental stresses. Thus, plants evolve in the direction of overcoming the environmental stresses. Some plant genes such as ARF, ABI3, NAC, HSF, and WRKY are known to respond to environmental stresses as they transcriptionally regulate the stress response pathways. For example, the OsWRKY76 gene contributes to the enhanced resistance to low temperatures and pathogenic infections. The AtWRKY28 also plays a role in environmental stresses. Zoysiagrass (Zoysia japonica Steud.) is popularly grown for gardens and golf courses. However, the function of the WRKY gene, another environmental stress-related gene, is not known in zoysiagrass. In this study, the ZjWRKY3 and ZjWRKY7 genes with one shared WRKY domain have been isolated in zoysiagrass. The expression of these genes increased in response to low temperature, drought, and salt stresses. Furthermore, the infection of the brown patch-causing Rhozoctonia solani induced the expression of ZjWRKY3 and ZjWRKY7. The corresponding proteins bind to the W-box of the Zjchi promoter, possibly regulating their transcriptions. The researchers suggest that the ZjWRKY3 and ZjWRKY7 genes transcriptionally regulate abiotic and biotic stress related downstream genes.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.381-388
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• 2009

On screening pathogen-resistant soybean, we identified a WRKY type transcription factor named a Glycine max WRKY1 (GmWRKY1). Expression of GmWRKY1 gene was induced in the soybean sprout by Pseudomonas infection. The GmWRKY1 was expressed in all of the tissues with high levels in stems, leaves and developing seeds. The protein Gm WRKY1 contains highly conserved two WRKY DNA-binding domains having two $C_2-H_2$ zinc-finger motif ($C-X_{4-5}-C-X_{22-23}-H-X-H$) in its N-terminal and C-terminal amino acid sequences. In electrophoresis mobility shift assay, the GmWRKY1 protein bound specifically to W-box elements in the promoters of defense related genes. These results demonstrated that GmWRKY1 is one of the soybean WRKY family genes and the plant-specific transcription factors for defense processes.

• Molecules and Cells
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• v.25 no.2
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• pp.196-204
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• 2008

Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsung-cho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to $F_2$ genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

• Molecules and Cells
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• v.22 no.1
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• pp.58-64
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• 2006

WRKY family proteins are a class of plant-specific transcription factors involved in stress response signaling pathways. In this study a gene encoding a putative WRKY protein was isolated from a pepper EST database (http://genepool.kribb.re.kr). The cDNA, named Capsicum annuum WRKY2 (CaWRKY2), encodes a putative polypeptide of 548 amino acids, containing two WRKY domains with zinc finger motifs and two potential nuclear localization signals. Northern blot analyses showed that CaWRKY2 mRNA was preferentially induced during incompatible interactions of pepper plants with PMMoV, Pseudomonas syringae pv. syringae 61, and Xanthomonas axonopodis pv. vesicatoria race 3. Furthermore, CaWRKY2 transcripts were strongly induced by wounding and ethephon treatment, whereas only moderate expression was detected following treatment with salicylic acid and jasmonic acid. CaWRKY2 was translocated to the nucleus when a CaWRKY2-smGFP fusion construct was expressed in onion epidermal cells. CaWRKY2 also had transcriptional activation activity in yeast. Taken together our data suggest that CaWRKY2 is a pathogen-inducible transcription factor that may have a role in early defense responses to biotic and abiotic stresses.

• Weed & Turfgrass Science
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• v.2 no.4
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• pp.368-375
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• 2013

Whole genome transcriptomes from Miscanthus species were sequenced and analyzed, which provided 50 different types of transcription factor (TF) involving various developmental processes or environmental stresses. Among the explored TF, WRKY gene family was the major type and one of the WRKY genes, MSIR7180_WRKY4, induced under low temperature environment was selected to investigate how the Miscanthus-originated MSIR7180_WRKY4 TF responds when exposed to low temperature in four warm-season turfgrasses (Z. matrella 'Semil', bermudagrass, St. Augustinegrass, and seashore paspalum). The MSIR7180_WRKY4 was expressed higher during low temperature period in Bermudagrass, but the expression was enhanced in St. Augustinegrass. In contrast, the gene in 'Semil' cultivar was barely expressed and relatively less expressed, but repressed gradually in seashore paspalum, which seems to allow two turfgrasses stay-green longer in the fall season. The results indicate that bermudagrass and St. Augustinegrass adapt to low temperature quickly, but relative tolerance to low or cold temperature at the molecular level needs to be further investigated at different physiological stages and the corresponding genes systematically.

• Molecules and Cells
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• v.37 no.7
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• pp.532-539
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• 2014

We isolated a rice (Oryza sativa L.) WRKY gene which is highly upregulated in senescent leaves, denoted OsWRKY42. Analysis of OsWRKY42-GFP expression and its effects on transcriptional activation in maize protoplasts suggested that the OsWRKY42 protein functions as a nuclear transcriptional repressor. OsWRKY42-overexpressing (OsWR KY42OX) transgenic rice plants exhibited an early leaf senescence phenotype with accumulation of the reactive oxygen species (ROS) hydrogen peroxide and a reduced chlorophyll content. Expression analysis of ROS producing and scavenging genes revealed that the metallothionein genes clustered on chromosome 12, especially OsMT1d, were strongly repressed in OsWRKY42OX plants. An OsMT1d promoter:LUC construct was found to be repressed by OsWRKY42 overexpression in rice protoplasts. Finally, chromatin immunoprecipitation analysis demonstrated that OsWRKY42 binds to the W-box of the OsMT1d promoter. Our results thus suggest that OsWRKY42 represses OsMT1d-mediated ROS scavenging and thereby promotes leaf senescence in rice.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.40-48
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• 2012

$Potato$ $virus$ $X$ (PVX) replication is precisely regulated by regulatory viral sequences and by viral and/or host proteins. In a previous study, we identified a 54-kDa cellular tobacco protein that bound to a region within the first 46 nucleotides (nt) of the 5' non-translated region (NTR) of the viral genome. Optimal binding was dependent upon the presence of an ACCA sequence at nt 10-13. To identify host factors that bind to 5' NTR elements including AC-rich sequences as well as stemloop 1 (SL1), we used northwestern blotting and matrixassisted laser desorption/ionization time-of-flight mass spectrometry for peptide mass fingerprinting. We screened several host factors that might affect PVX replication and selected a candidate protein, $Nicotiana$ $tabacum$ WRKY transcription factor 1 (NtWRKY1). We used a $Tobacco$ $rattle$ $virus$ (TRV)-based virus-induced gene silencing (VIGS) system to investigate the role of NtWRKY1 in PVX replication. Silencing of $WRKY1$ in $Nicotiana$ $benthamiana$ caused lethal apical necrosis and allowed an increase in PVX RNA accumulation. This result could reflect the balancing of PVX accumulation in a systemic $N.$ $benthamiana$ host to maintain PVX survival and still produce a suitable appearance of mosaic and mottle symptoms. Our results suggest that PVX may recruit the WRKY transcription factor, which binds to the 5' NTR of viral genomic RNA and acts as a key regulator of viral infection.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2004.10a
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• pp.62-65
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• 2004

To protect the watermelon against soil-borne pathogens, we are currently producing disease-resistant transgenic root stock for the growth of watermelon, A defensin gene (J1-1) from Capsicum annum, a ACC deaminase gene from Pseudomonas syringae, a galactinol synthase (CsGolS) gene from Cucumis sativus, and a WRKY (CvWRKY2) gene from Citullus vulgaris were used as transgenes for disease resistance. The gene were transformed into a inbred line (6-2-2) of watermelon, Kong-dae watermelon and a inbred line (GO702S) of gourd, respectively, by Agrobacterium-mediated transformation. Putative transgenic plants were selected in medium containing 100mg/L kanamycin, and then integration of the genes into the genomic DNA were demonstrated by PCR analysis. Successful integration of the gene in regenerated plants was also confirmed by PCR (Figf 1), genomic Southern blot (Fig 2), RT-PCR (Fig 3), and Northern blot analysis(Fig 4). Several T1 lines having different transgene were produced, and disease resistance of the T1 lines are under estimation.

• Plant Biotechnology Reports
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• v.4 no.1
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• pp.29-35
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• 2010

NPR1 is a positive regulator of systemic acquired resistance in Arabidopsis and rice. Expression of the rice gene OsNPR1 is induced by salicylic acid (SA). To identify the region of the OsNPR1 promoter involved in response to SA, we carried out deletion mutagenesis of the region 1005 bp upstream of the OsNPR1 start codon. Ciselement analysis revealed that the OsNPR1 promoter contains W-boxes and ASF1 motifs, both of which are known to be functional cis-elements of the WRKY and bZIP proteins, respectively. The deletion constructs 1005:LUC and 752:LUC, were induced by up to 4.3- and 3.8-fold, respectively, following SA treatment, suggesting that W-boxes and ASF1 motifs may play an important role in the strong induction of these constructs by SA. Using mutation analysis, we also showed that both the W-box and ASF1 motif are necessary for SA-induced expression of OsNPR1.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.232-232
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• 2022

We reported in our recent studies that the combined treatment of Paenibacillus yonginensis DCY84^T (DCY84^T) and Silicon (Si) promotes initial plant growth and increases resistance to biotic and abiotic stress. To understand the molecular background of these phenotypes, Liquid Chromatography Mass Spectrometry (LC-MS) analysis was performed, and it was confirmed that unsaturated fatty acid metabolites such as oleic acid and linoleic acid decreased in response to the combined treatment of DCY84^T and Si. The stearoyl-acyl carrier protein desaturase (SACPD) introduces the cis double bond into the acyl-ACPs at C9, resulting in the production of unsaturated fatty acid. We identified OsSSI2 encoding SACPD in rice and found that the expression of OsSSI2 was reduced under DCY84^T and Si treatment. Furthermore, qRT-PCR analysis revealed that the expression of OsWRKY45, which is downstream of OsSSI2, was upregulated in response to DCY84^T and Si treatment. These results enable the speculation that activation of the salicylic acid (SA)-responsive gene, OsWRKY45, may contribute to enhancing biological stress resistance. Based on this, we propose a probable model for the rice defense pathway following DCY84^T and Si treatment. This model retains a WRKY45-dependent but NH1(NPR1)-independent SA signaling pathway.