Sang Ryeol Park

Expression of StMYB1R-1, a Novel Potato Single MYB-like domain Transcription Factor, Increases Drought Tolerance

Potato (Solanum tuberosum) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms for such stresses in potato are largely unknown. To identify stress-related factors in potato, we previously carried out a genetic screen of potato plants exposed to abiotic environmental stress conditions using reverse northern-blot analysis. A cDNA encoding a putative R1-type MYB-like transcription factor (StMYB1R-1) was identified as a putative stress-response gene. Here, the transcript levels of StMYB1R-1 were enhanced in response to several environmental stresses in addition to drought but were unaffected by biotic stresses. The results of intracellular targeting and quadruple 9-mer protein-binding microarray analysis indicated that StMYB1R-1 localizes to the nucleus and binds to the DNA sequence G/AGATAA. Overexpression of a StMYB1R-1 transgene in potato plants improved plant tolerance to drought stress while having no significant effects on other agricultural traits. Transgenic plants exhibited reduced rates of water loss and more rapid stomatal closing than wild-type plants under drought stress conditions. In addition, overexpression of StMYB1R-1 enhanced the expression of drought-regulated genes such as AtHB-7, RD28, ALDH22a1, and ERD1-like. Thus, the expression of StMYB1R-1 in potato enhanced drought tolerance via regulation of water loss. These results indicated that StMYB1R-1 functions as a transcription factor involved in the activation of drought-related genes.

Molecular characterization of Oryza sativa WRKY6, which binds to W-box-like element 1 of the Oryza sativa pathogenesis-related (PR) 10a promoter and confers reduced susceptibility to pathogens

WRKY proteins are transcription factors (TFs) that regulate the expression of defense-related genes. The salicylic acid (SA)-inducible Oryza sativa WRKY6 (OsWRKY6) was identified as a positive regulator of Oryza sativa pathogenesis-related 10a (OsPR10a) by transient expression assays. A physical interaction between OsWRKY6 and W-box-like element 1 (WLE1), which positively regulates OsPR10a/probenazole induced protein 1 expression, was verified in vitro. Several pathogenesis-related (PR) genes were constitutively activated, including OsPR10a, and transgenic rice (Oryza sativa) plants overexpressing (ox) OsWRKY6 exhibited enhanced disease resistance to pathogens. By contrast, PR gene induction was compromised in transgenic OsWRKY6-RNAi lines, suggesting that OsWRKY6 is a positive regulator of defense responses. OsWRKY6-ox lines displayed leaf lesions, and increased OsWRKY6 levels caused cell death. Salicylic acid (SA) concentrations were higher in OsWRKY6-ox lines than in wild-type (WT) plants, and transcript levels of Oryza sativa isochorismate synthase 1 (OsICS1), which encodes a major enzyme involved in SA biosynthesis, were higher in OsWRKY6-ox lines than in WT. OsWRKY6 directly bound to the OsICS1 promoter in vivo. This indicates that OsWRKY6 can directly regulate OsICS1 expression and thereby increase SA concentrations. OsWRKY6 autoregulates its own expression. OsWRKY6 protein degradation is possibly regulated by ubiquitination. Our results suggest that OsWRKY6 positively regulates defense responses through activation of OsICS1 expression and OsWRKY6 stabilization.

Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

Identification of AtWRKY75 as a transcriptional regulator in the defense response to Pcc through the screening of Arabidopsis activation-tagged lines

The necrotrophic pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc) causes soft rot in a broad range of plant hosts. Approximately 60,000 independent seeds from Arabidopsis activation tagging lines were inoculated with Pcc and screened for resistant mutants. An Rpe1 (resistance protein to Pectobacterium 1) mutant, which had more resistance to Pcc than wild-type (WT) plants, was selected for further study. The T-DNA inserting locus in Rpe1 was located on the middle of chromosome V by flanking sequence analysis. Through expression analysis with several genes adjacent to the T-DNA tagging region, AtWRKY75 gene was highly up-regulated in the Rpe1 mutant compared to the WT plant. The up-regulation of AtWRKY75 gene was shown to be correlated on the induction of the PDF1.2, VSP1 and PR1 genes compared to the WT plant. AtWRKY75 over-expression lines exhibited reduced Pcc bacterial growth compared to WT. Taken together, our data suggest that AtWRKY75 should be a positive regulator in the JA- or SA-mediated defense signaling responses to Pcc.

Overexpression of the Brassica rapa transcription factor WRKY12 results in reduced soft rot symptoms caused by Pectobacterium carotovorum in Arabidopsis and Chinese cabbage

Chinese cabbage (Brassica rapa L. ssp. pekinensis), an important vegetable crop, can succumb to diseases such as bacterial soft rot, resulting in significant loss of crop productivity and quality. Pectobacterium carotovorum ssp. carotovorum (Pcc) causes soft rot disease in various plants, including Chinese cabbage. To overcome crop loss caused by bacterial soft rot, a gene from Chinese cabbage was isolated and characterised in this study. We isolated the BrWRKY12 gene from Chinese cabbage, which is a group II member of the WRKY transcription factor superfamily. The 645-bp coding sequence of BrWRKY12 translates to a protein with a molecular mass of approximately 24.4 kDa, and BrWRKY12 was exclusively localised in the nucleus. Transcripts of BrWRKY12 were induced by Pcc infection in Brassica. Heterologous expression of BrWRKY12 resulted in reduced susceptibility to Pcc but not to Pseudomonas syringae pv. tomato in Arabidopsis. Defence-associated genes, such as AtPDF1.2 and AtPGIP2, were constitutively expressed in transgenic lines overexpressing BrWRKY12. The expression of AtWKRY12, which is the closest orthologue of BrWRKY12, was down-regulated by Pcc in Arabidopsis. However, the Atwrky12-2 mutants did not show any difference in response to Pcc, pointing to a difference in function of WRKY12 in Brassica and Arabidopsis. Furthermore, BrWRKY12 in Chinese cabbage also exhibited enhanced resistance to bacterial soft rot and increased the expression of defence-associated genes. In summary, BrWRKY12 confers enhanced resistance to Pcc through transcriptional activation of defence-related genes.

The C-terminal region of OsWRKY30 is sufficient to confer enhanced resistance to pathogen and activate the expression of defense-related genes

WRKY proteins are known to play major roles in defense signaling. We identified a pathogen-inducible and SA-inducible OsWRKY30. Our cDNA clone encodes the C-terminal region (CTR) of OsWRKY30. CTR-OsWRKY30 includes the C-terminal WRKY domain and nuclear localization sequence. CTR-OsWRKY30 was sufficient to bind W-box sequences (TTGACC/T). Over-expression of the CTR-OsWRKY30 resulted in enhanced resistance to pathogens in Arabidopsis and rice. Defense-related genes were constitutively expressed in transgenic Arabidopsis and rice over-expressing CTR-OsWRKY30. Based on promoter transient assays, CTR-OsWRKY30 is sufficient to activate OsPR10a promoter as much as full length OsWRKY30. Taken together, CTR-OsWRKY30 positively regulates defense signaling, thereby resulting in enhanced resistance to pathogens.

Rice WRKY11 Plays a Role in Pathogen Defense and Drought Tolerance

BackgroundPlants are frequently subjected to abiotic and biotic stresses, and WRKY proteins play a pivotal role in the response to such stress. OsWRKY11 is induced by pathogens, drought, and heat, suggesting a function in biotic and abiotic stress responses.ResultsThis study identified OsWRKY11, a member of WRKY group IIc. It is a transcriptional activator that localized to the nucleus. Ectopic expression of OsWRKY11 resulted in enhanced resistance to a bacterial pathogen, Xanthomonas oryzae pv. oryzae; resistance was compromised in transgenic lines under-expressing OsWRKY11. Ectopic expression of OsWRKY11 resulted in constitutive expression of defense-associated genes, whereas knock-down (kd) of OsWRKY11 reduced expression of defense-associated genes during pathogen attack, suggesting that OsWRKY11 activates defense responses. OsWRKY11 bound directly to the promoter of CHITINASE 2, a gene associated with defense, and activated its transcription. In addition, ectopic expression of OsWRKY11 enhanced tolerance to drought stress and induced constitutive expression of drought-responsive genes. Induction of drought-responsive genes was compromised in OsWRKY11-kd plants. OsWRKY11 also bound directly to the promoter of a drought-responsive gene, RAB21, activating its transcription. In addition, OsWRKY11 protein levels were controlled by the ubiquitin-proteasome system.ConclusionOsWRKY11 integrates plant responses to pathogens and abiotic stresses by positively modulating the expression of biotic and abiotic stress-related genes.

Overexpression of rice NAC transcription factor OsNAC58 on increased resistance to bacterial leaf blight

Bacterial blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) greatly reduces the growth and productivity of this important food crop. Therefore, we sought to increase the resistance of rice to bacterial blight by using a NAC (NAM, ATAF, and CUC) transcription factor, one of the plant-speci...

Selection of a Susceptible Line (Susceptible to Pectobacterium 1, Atstp1) to Soft-rot Disease in T-DNA Insertion Mutants Pool of Arabidopsis

Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft rot disease in various plants. Although many studies about Pcc have been going on, little is known yet about the defense genes from plants. To identify defense associated genes in response to Pcc, we screened about 20 thousand Arabidopsis T-DNA knock out lines by inoculation with Pcc. We obtained a line (Atspt1) showing more susceptible symptom compared to WT (Col-0) on 1 day after the inoculation of Pcc on leaves of Arabidopsis with toothpicks. In this study, we optimized the system to select resistant and susceptible lines to Pcc from T-DNA inserted pool of Arabidopsis and expect the system and Atspt1 might be used for molecular breeding to produce resistant vegetables against Pcc.

Isolation and Characterization of Rice OsHRL Gene Related to Bacterial Blight Resistance

Department of Environmental Horticulture, University of Seoul, Seoul 130-743, Korea(Received on June 21, 2010; Accepted on November 2, 2010)The expression of HR-like lesion inducing gene of Oryzasativa (OsHRL) was slightly increased by Xanthomonasoryzae pv. oryzae (Xoo) infection. Transgenic rice plantsover-expressing OsHRL gene were challenged with Xooand the development of disease symptoms were examin-ed to investigate the effect of OsHRL gene expression onplant defense responses. The over-expression of OsHRLincreased disease resistance against Xoo compared withwild type plants. Keywords :avrBs2 homologue, OsHRL, rice, Xanthomonasoryzae pv. oryzaeRice is one of the most important crop species, and its yieldis constantly affected by several major diseases such asbacterial blight, blast, sheath blight, and tungro (Dai et al.,2007). Bacterial blight, caused by Xanthomonas oryzae pv.oryzae (Xoo), is a serious agronomic problem in many rice-growing regions (Mew et al., 1993). Therefore, under-standing of molecular mechanisms underlying host resis-tance to pathogens is essential to develop better controllingstrategies against rice diseases. Because of availability ofthe genome sequences, rice (Sasaki et al., 2005) and Xoo(Lee et al., 2005) are considered as a model system of plantand bacterial pathogen, respectively (Li et al., 2006). It has been previous reported that the avrBs2 gene, whichwas identified in many species of Xanthomonas, encodesagrocinopine synthase and glycerol phosphodiesterase(Swords et al. 1996). The avrBs2 of Xanthomonas cam-pestris pv. vesicatoria (Xcv) is specifically recognized bypepper Bs2 resulting in localized cell death and plantresistance (Mudgett et al., 2000). We thus attempt to iden-tify avrBs2-interacting partner from rice. AvrBs2 (Kearneyand Staskawicz, 1990; Swords et al., 1996) homologue wasselected from Xoo strain KACC10859 (K1 race), based ongenomic sequences of Xoo strain KACC10331 (K1 race,Lee et al., 2005). Xoo strain KACC10859 harbors singlecopy gene of avrBs2 which is used as bait to isolate inter-acting host components using yeast two-hybrid screening.Possible involvement in plant immunity of one selectedgene, OsHRL encoding HR-like lesion inducing protein,was evaluated. Rice (O. sativa L. cv. Dongjin) seedlings were grown for6 weeks in a greenhouse under controlled temperature (20-30°C) and natural light conditions. For bacterial inocula-tions, Xoo strain was grown in Peptone Sucrose medium at28°C (Karganilla et al., 1973). The cells were collected bycentrifugation, and diluted to OD