Woobong Choi

Molecular Cloning and Functional Analysis of Rice (Oryza sativa L.) OsNDR1 on Defense Signaling Pathway

A novel rice (Oryza sativa L.) gene, homologous to Arabidopsis pathogenesis-related NDR1 gene, was cloned from cDNA library prepared from 30 min Magnaporthe grisea -treated rice seedling leaves, and named as OsNDR1. OsNDR1 encoded a 220-aminoacid polypeptide and was highly similar to the Arabidopsis AtNDR1 protein. OsNDR1 is a plasma membrane (PM)-localized protein, and presumes through sequence analysis and protein localization experiment. Overexpression of OsNDR1 promotes the expression of PBZ1 that is essential for the activation of defense/stressrelated gene. The OsNDR1 promoter did not respond significantly to treatments with either SA, PBZ, or ETP. Exogenously applied BTH induces the same set of SAR genes as biological induction, providing further evidence for BTH as a signal. Presumably, BTH is bound by a receptor and the binding triggers a signal transduction cascade that has an ultimate effect on transcription factors that regulate SAR gene expression. Thus OsNDR1 may act as a transducer of pathogen signals and/or interact with the pathogen and is indeed another important step in clarifying the component participating in the defense response pathways in rice.

Genome-scale analysis of ABC transporter genes and characterization of the ABCC type transporter genes in Magnaporthe oryzae

Rapid adaptation to various environmental stresses is a prerequisite for successful infection in fungal pathogens. ABC transporters are responsible for regulating intracellular levels of cytotoxic or xenobiotic compounds, suggesting a crucial role in pathogenesis. Here, we report genome-scale identification of putative ABC transporter genes in Magnaporthe oryzae. A total of 50 ABC transporter genes were predicted and phylogenetic analysis divided them into 11 subfamily groups: ABCA, ABCB, ABCC-1, ABCC-2, ABCD, ABCE, ABCF, ABCG-1, ABCG-2, ABCI, and YDR061W-like. In the 11 ABCC subfamily genes, the transcript levels were elevated during infection stages and after exposure to various abiotic stresses. Based on expression pattern, three representative genes, MoABC5, MoABC6 and MoABC7, were selected. Functional analysis of MoABC5, MoABC6 and MoABC7 revealed that the genes may be responsible for virulence, abiotic stress tolerance, and conidiation, respectively. Our data will be providing valuable information to examine the role of ABC transporter genes in M. oryzae.

Analyses of the Less Benzimidazole-sensitivity of the Isolates of Colletotrichum spp. Causing the Anthracnose in Pepper and Strawberry

The anthracnose disease on pepper fruits in Korea was caused by Colletotrichum acutatum as well as C. gloeosporioides. Since C. acutatum showed less sensitivity to benomyl, it was analyzed whether the less sensitivity was given by the same mechanism for the fungicide resistance of C. gloeosporioides. The isolates of C. acutatum were less sensitive to the three benzimidazole fungicides tested, benomyl, carbendazim, and thiophanate-methyl. However, the of C. acutatum isolates were different from the resistant isolates of C. gloeosporioides in their response to diethofencarb, one of N-phenyl-carbamates; the former was still less sensitive to diethofencarb than the latter. The differences in the resistance mechanisms in two species were conspicuous in sequence analysis of the tub2 genes. The genes from C. acutatum did not show any non-synonymous base substitutions at the regions known to be correlated with the benzimidazole-resistance. All of these data may indicate that the less sensitivity of C. acutatum to benomyl is based on different mechanism(s) from that of C. gloeosporioides.

Antifungal Activity of Paenibacillus kribbensis Strain T-9 Isolated from Soils against Several Plant Pathogenic Fungi

The bacterial strain T-9, which shows strong antifungal activity, is isolated from the soils of Samcheok, Gangwondo and identified as Paenibacillus kribbensis according to morphological and taxonomic characteristics and 16S rRNA gene sequence analysis. The P. kribbensis strain T-9 strongly inhibits the growth of various phytopathogenic fungi including Botrytis cinerea, Colletotricum acutatum, Fusarium oxysporum f. sp. radicis-lycopersici, Magnaporthe oryzae, Phytophthora capsici, Rhizoctonia solani, and Sclerotium cepivorum in vitro. Also, the P. kribbensis strain T-9 exhibited similar or better control effects to plant diseases than in fungicide treatment through in vivo assays. In the 2-year greenhouse experiments, P. kribbensis strain T-9 was highly effective against clubroot. In the 2-year field trials, the P. kribbensis strain T-9 was less effective than the fungicide, but reduced clubroot on Chinese cabbage when compared to the control. The above-described results indicate that the strain T-9 may have the potential as an antagonist to control various phytopathogenic fungi.

Astaxanthin Improves Stem Cell Potency via an Increase in the Proliferation of Neural Progenitor Cells

The present study was designed to investigate the question of whether or not astaxanthin improves stem cell potency via an increase in proliferation of neural progenitor cells (NPCs). Treatment with astaxanthin significantly increased proliferation and colony formation of NPCs. For identification of possible activated signaling molecules involved in active cell proliferation occurring after astaxanthin treatment, total protein levels of several proliferation-related proteins, and expression levels of proliferation-related transcription factors, were assessed in NPCs. In Western blot analysis, astaxanthin induced significant activation of phosphatidylinositol 3-kinase (PI3K) and its downstream mediators in a time-dependent manner. Results of RT-PCR analysis showed upregulation of proliferation-related transcription factors and stemness genes. To estimate the relevance of PI3K and mitogen-activated protein, or extracellular signal-regulated kinase kinase (MEK) signaling pathways in cell growth of astaxanthin-treated NPCs, inhibition assays were performed with LY294002, a specific inhibitor of PI3K, and PD98059, a specific inhibitor of MEK, respectively. These results clearly showed that astaxanthin induces proliferation of NPCs via activation of the PI3K and MEK signaling pathways and improves stem cell potency via stemness acting signals.

Fusarium Wilt of Winter Daphne (Daphne odora Thunb.) Caused by Fusarium oxysporum

Severe wilt disease epidemic was found on winter daphnes (Daphne odora Thunb.) cultivated in farmers, nurseries in Suncheon, Jeonnam in 2003. Typical symptoms appeared on the leaves of winter daphne as yellowish wilts and turned brown from the lower leaves on the same plant. Severely infected leaves were defoliated, resulting in blight of stems and eventual death of the entire plant. Black decayed vascular tissues were distinctly observed in a wilted plant. Fusarium sp. was isolated from the diseased plants repeatedly and its pathogenicity was confirmed by artificial inoculation on healthy plants. The fungus was identified as Fusarium oxysporum on the basis of the morphological and cultural characteristics on potato dextrose agar and carnation leaf agar. The optimum temperature for fungal growth was around and the fungal growth was inhibited by metconazole, triflumizole and trifloxystrobin on potato dextrose agar. This is the first report on the wilt disease of winter daphnes caused by F.oxysporum in Korea.

Identification of Genes Expressed during Conidial Germination of the Pepper Anthracnose Pathogen, Colletotrichum acutatum

Genes expressed during conidial germination of the pepper anthracnose fungus Colletotrichum acutatum were identified by sequencing the 5’ end of unidirectional cDNA clones prepared from the conidial germination stage. A total of 983 expressed sequence tags (ESTs) corresponding to 464 genes, 197 contigs and 267 singletons, were generated. The deduced protein sequences from half of the 464 genes showed significant matches (e value less than 10-5) to proteins in public databases. The genes with known homologs were assigned to known functional categories. The most abundantly expressed genes belonged to those encoding the elongation factor, histone protein, ATP synthease, 14-3-3 protein, and clock controlled protein. A number of genes encoding proteins such as the GTP-binding protein, MAP kinase, transaldolase, and ABC transporter were detected. These genes are thought to be involved in the development of fungal cells. A putative pathogenicity function could be assigned for the genes of ATP citrate lyase, CAP20 and manganese-superoxide dismutase.

Sequence and expression analysis of extracellular signal-regulated kinase 1 from flounder (Paralichthys olivaceous)

Extracellular signal-regulated kinases (ERKs) are a subgroup of mitogen-activated protein kinases (MAPK) that function as important intermediates in signal transduction pathways initiated by several types of cell surface receptors. We cloned a transcript of ERK1 from a cDNA library of flounder leukocytes stimulated with bacterial lipopolysaccharide and hemagglutinin lectin. Flounder ERK1 consists of 1502 nucleotides and encodes a polypeptide of 393 amino acids. Flounder ERK1 showed 90 and 89% amino acid sequence identity to ERK1 of carp and zebrafish, respectively, and over 85% to that of mammals. Multiple bands were detected by Southern blot analysis of flounder genomic DNA after digestion with various restriction enzymes, implying the presence of additional MAPK genes in flounder. Real-time PCR revealed the ubiquitous expression of flounder MAPK in all tissues with high levels of transcription in brain, gill, and fin, but not in muscle or skin. Flounder MAPK was successfully expressed in mammalian COS1 cells and phosphorylated myelin basic protein (MBP) substrate when the cells were stimulated with PMA or EGF, indicating that flounder MAPK is functional in animal cells.

Characterization of the Chaetoceros salsugineum nuclear inclusion virus coat protein gene

Analysis of the genome of Chaetoceros salsugineum nuclear inclusion virus (CsNIV) revealed the presence of six putative open reading frames (ORFs) in the genome. We further characterized ORF3, which encodes a putative coat protein. Polymerase chain reaction (PCR) using ORF3 gene-specific primers amplified a single DNA band nearly 1.2kb. This amplified product was gel-purified, cloned, sequenced, and expressed in Escherichia coli. Specific antiserum was raised against the recombinant protein and used for Western blotting to test whether the ORF3 protein is the CsNIV coat protein. One major CsNIV protein of approximately 46kDa reacted positively with the antiserum, suggesting that this antiserum is specific for the CsNIV coat protein. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the 46kDa structural band revealed 14 peptide sequences that matched the ORF3 regions of CsNIV. The expression of ORF3 in host cells was examined by constructing a cDNA library of CsNIV-infected cells. Nucleotide sequences of the cDNA clones were complementary to various regions of both CsNIV ORF3 and ORF4; however, no clones containing only the ORF3 region were identified. Also, Northern blotting revealed a single 2.5-kb transcript, indicating that ORF3 could be transcribed together with ORF4.

Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1.

Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5′-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.