Shin-Chul Bae

Genes up-regulated during red coloration in UV-B irradiated lettuce leaves

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3′,5′-hydroxylase (F3′,5′H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.

Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

Expression analysis of rice VQ genes in response to biotic and abiotic stresses

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Proteins containing a short VQ (FxxxVQxLTG) motif have been recently shown to interact with WRKY transcription factors, implying that AtVQ proteins are important in the plant defense responses in Arabidopsis, either as positive or negative cofactors of WRKY transcription factors. Thirty-nine Oryza sativa genes containing the VQ motif (OsVQs) were identified and the genome structures of OsVQ proteins were characterized through genome-wide analysis in rice. Also, phylogenetic tree analysis was performed with the VQ domain of Arabidopsis and rice. The expression patterns of these OsVQ genes in plants under several stress treatments were assessed, specifically, following infection with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), treatment with abscisic acid (ABA), or exposure to drought. The cellular localization of a few OsVQ proteins was examined using rice protoplast system. Based on our results, we suggest that OsVQ proteins function as important co-regulators during the plant defense response to biotic and abiotic stresses.

Priming by Rhizobacterium Protects Tomato Plants from Biotrophic and Necrotrophic Pathogen Infections through Multiple Defense Mechanisms

A selected strain of rhizobacterium, Pseudomonas putida strain LSW17S (LSW17S), protects tomato plants (Lycopersicon esculentum L. cv. Seokwang) from bacterial speck by biotrophic Pseudomonas syringae pv. tomato strain DC3000 (DC3000) and bacterial wilt by necrotrophic Ralstonia solanacearum KACC 10703 (Rs10703). To investigate defense mechanisms induced by LSW17S in tomato plants, transcription patterns of pathogenesis-related (PR) genes and H2O2 production were analyzed in plants treated with LSW17S and subsequent pathogen inoculation. LSW17S alone did not induce transcriptions of employed PR genes in leaves and roots. DC3000 challenge following LSW17S triggered rapid transcriptions of PR genes and H2O2 production in leaves and roots. Catalase infiltration with DC3000 attenuated defense-related responses and resistance against DC3000 infection. Despite depriving H2O2 production and PR1b transcription by the same treatment, resistance against Rs10703 infection was not deterred significantly. H2O2 is indispensable for defense signaling and/or mechanisms primed by LSW17S and inhibition of bacterial speck, however, it is not involved in resistance against bacterial wilt.

Evaluation of bakanae disease progression caused by Fusarium fujikuroi in Oryza sativa L.

Bakanae disease caused by Fusarium fujikuroi is an important fungal disease in rice. Among the seven strains isolated from symptomatic rice grains in this study, one strain, FfB14, triggered severe root growth inhibition and decay in the crown and root of rice seedlings. The remaining six strains caused typical Bakanae symptoms such as etiolation and abnormal succulent rice growth. To reveal the relationship between mycelial growth in the infected tissues and Bakanae disease progression, we have established a reliable quantification method using real time PCR that employs a primer pair and dual-labeled probe specific to a unigene encoding F. fujikuroi PNG1 (FfPNG1), which is located upstream of the fumonisin biosynthesis gene cluster. Plotting the crossing point (CP) values from the infected tissue DNAs on a standard curve revealed the active fungal growth of FfB14 in the root and crown of rice seedlings, while the growth rate of FfB20 in rice was more than 4 times lower than FfB14. Massive infective mycelial growth of FfB14 was evident in rice stems and crown; however, FfB20 did not exhibit vigorous growth. Our quantitative evaluation system is applicable for the identification of fungal virulence factors other than gibberellin.

OsWRKY51, a rice transcription factor, functions as a positive regulator in defense response against Xanthomonas oryzae pv. oryzae

Key messageOsWRKY51 functions as a positive transcriptional regulator in defense signaling againstXanthomonas oryzae pv. oryzaeby direct DNA binding to the promoter of defense related gene, OsPR10a.AbstractOsWRKY51 in rice (Oryza sativa L.) is induced by exogenous salicylic acid (SA) and inoculation with Xanthomonas oryzae pv. oryzae (Xoo). To examine the role of OsWRKY51 in the defense response of rice, we generated OsWRKY51 overexpressing and underexpressing transgenic rice plants. OsWRKY51-overexpressing transgenic rice lines were more resistant to Xoo and showed greater expression of defense-related genes than wild-type (WT) plants, while OsWRKY51-underexpressing lines were more susceptible to Xoo and showed less expression of defense-associated genes than WT plants. Transgenic lines overexpressing OsWRKY51 showed growth retardation compared to WT plants. In contrast, transgenic lines underexpressing OsWRKY51 by RNA interference showed similar plant height with WT plants. Transient expression of OsWRKY51-green fluorescent protein fusion protein in rice protoplasts revealed that OsWRKY51 was localized in the nucleus. OsWRKY51 bound to the W-box and WLE1 elements of the OsPR10a promoter. Based on these results, we suggest that OsWRKY51 is a positive transcriptional regulator of defense signaling and has direct DNA binding ability to the promoter of OsPR10a, although it is reported to be a negative regulator in GA signaling.

Arabidopsis Cell Death in Compatible and Incompatible Interactions with Alternaria brassicicola

Two strains of necrotrophic Alternaria brassicicola, Ab40857 and Ab42464, are virulent on Korean cabbage and several wild types of Arabidopsis thaliana. Interaction between Ab42464 and Col-0 was compatible, whereas interaction between Ab40857 and Col-0 was incompatible. The loss of defense, no death (dnd) 1 function abrogated the compatibility between Ab42464 and Col-0, and the accelerated cell death (acd) 2 mutation attenuated the Col-0’s resistance against Ab40857. These two fungal strains induced PR1 transcription in Col-0. Ab40857 accelerated transcription of PDF1.2, THI2.1, CAT, and POX by 12 h compared to those challenged with Ab42464. More abundant cell death was observed in Col-0 infected with Ab42464, however, callose deposition was evident in the incompatible interaction. Remarkably, Ab40857-infected areas of acd2-2 underwent rampant cell death and Ab42464 triggered callose production in dnd1-1. Furthermore, the incompatibility between Ab40857 and Col-0 was nullified by the coronatine-insensitive 1 (coi1) and phytoalexin-deficient 3 (pad3) mutations but not by nonexpresser of PR genes (npr1) and pad4. Ab40857 induced abundant cell death in pad3. Taken together, cell death during the early infection stage is a key determinant that discriminates between a compatible interaction and an incompatible one, and the resistance within Col-0 against Ab40857 is dependent on a defense-signaling pathway mediated by jasmonic acid and PAD3.

Isolation and Expression Analysis of Brassica rapa WRKY 7

Brassica Genomics Team, National Institute of Agricultural Biotechnology (NIAB), RDA, Suwon, Korea (Received on August 11, 2008; Accepted on November 5, 2008)The cDNA clone of Brassica rapa WRKY7 (BrWRKY7)was obtained from EST collection in Brassica genomicsteam and its DNA sequence was determined. The cDNAclone is 1,037 bp long in nucleotides and encodes anopen reading frame of 307 amino acids. Based on aphylogenetic tree, BrWRKY7 belongs to group IId.BrWRKY7 was induced by wound and SA. It was alsoinduced by pathogen attack such as Xanthomonascampestris pv. campestris (Xcc), suggesting that thisBrWRKY may play an essential role in defense responseof chinese cabbages. Keywords : Brassica rapa, WRKY transcription factor Plants have a variety of active defense mechanisms toprotect themselves from pathogen infection. Plant defenseresponses result from the transcriptional activation of alarge number of genes upon pathogen infection or treatmentwith pathogen elicitors (Ruston and Somssich, 1998;Schreiber and Desveaux, 2008; Yang et al., 1997). MostWRKY genes are rapidly induced by pathogens, pathogenelicitors, or salicylic acid (SA) treatment in mostly Arabi-dopsis and rice (Chen and Chen, 2000; Dong et al., 2003;Eulgem et al., 1999; Ryu et al., 2006). Therefore, it hasbeen implicated that WRKY proteins are major transcrip-tional regulators in defense signaling (Chen and Chen,2000, 2002; Chen etal., 2002; Cormack et al., 2002;Deslandes et al., 2002; Du and Chen, 2000; Eulgem et al.,1999; Kim et al., 2000; Liu et al., 2005, 2006; Maleck et al.,2000; Robatzek and Somssich, 2001, 2002; Yoda et al.,2002; Yu et al., 2001). In this study, based on globalexpression profiling analysis of Brassica rapa genes on25K cDNA oligomer chip, BrWRKY7 was isolated andcharacterized.Chinese cabbage (Brassica rapa ssp. pekiness cv. chiibu)was grown in a greenhouse for 3 weeks. Three-week oldchinese cabbage seedlings were treated with salicylic acid(SA) at 1 mM or buffer, respectively. Chinese cabbageleaves were harvested at the times indicated in the figures.For bacterial inoculations, a compatible strain of Xcc toBrassica rapa ssp. pekiness cv. chiibu was grown in PSA

Quantification of Alternaria brassicicola infection in the Arabidopsis thaliana and Brassica rapa subsp. pekinensis.

Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.

Screening of GLA (γ-Linolenic Acid) from Fungi by Gas Chromatography and Mass Spectroscopy

In order to select acid (GLA)-producing fungi, a total of forty-four strains of 4 genera such as Phytophthora, Pythium, Mucor and Rhizopus were obtained from Koran Agricultural Culture Collection (KACC) and then analysed by using GC-FID and GC-MS. GLA was detected on 39 fungal strains, and the highest rate of GLA was found as 24.8% of total fatty acids on Mucor hiemalis f. sp. hiemalis KACC 40264. Total GLA content of Zygomycota was comparatively high - Mucor (14.2%) and Rhizopus (14.3%), whereas that of Oomycetes was low - Phytophthora (3.3%) and Pythium (3.0%). Moreover, total fatty acids of the Zygomycota fungi such as Mucor (15.4 mg/100 ml) and Rhizopus (7.1 mg/100 ml) were higher compared with the Oomycetes such as Phytophthora (2.6 mg/100 ml) and Pythium (4.5 mg/100 ml). Thus, two genera such as Mucor and Rhizopus have higher potential as an useful microbial resource. The total fatty acid content varies even within the strains of the same genus e.g. Mucor. M. blumbeus KACC 40935 showed the highest values on productivity (18.2%) of GLA and total fatty acid contents (50.8 mg/100 ml liquid medium).