Phytophthora species, new threats to the plant health in Korea.

Given the lack of a resistant genetic pool in host plants, the introduction of exotic invasive pathogens can result in epidemics that affect a specific ecosystem and economy. Plant quarantine, which is designed to protect endemic plant resources, is a highly invaluable safeguard that should keep biosecurity with increasing international trade and global transportation. A total of 34 species of plant pathogens including Phytophthora infestans were documented as introduced from other countries into Korea from 1900 to 2010. The genus Phytophthora, classified in oomycetes, includes more than 120 species that are mostly recognized worldwide as highly invasive plant pathogens. After 2000, over 50 new species of Phytophthora were identified internationally as plant pathogens occurring in crops and forest trees. In Korea, Phytophthora is also one of the most serious plant pathogens. To date, 22 species (about one-fifth of known species) of the genus have been identified and reported as plant pathogens in the country. The likelihood of new exotic Phytophthora species being introduced into Korea continues to increase, thus necessitating intensive plant quarantine inspections. As new potential threats to plant health in Korea, six Phytophthora species, namely, P. alni, P. inundata, P. kernoviae, P. pinifolia, P. quercina, and P. ramorum, are discussed in this review with focus on history, disease, biology, management, and plant quarantine issues.

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.

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.

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.

Seed-borne Brachycladium penicillatum Intercepted under Plant Quarantine Inspection in Korea.

A fungus detected from the importing seeds of Papaver rhoeas under plant quarantine inspection in Korea was identified as Brachycladium penicillatum Corda. It differed in morphological characteristics from a similar species, B. papaveris, which was known to form no macroconidiophores and no microsclerotia. Since the first interception in 2006, this fungus has frequently been found from importing seeds of Papaver spp. It was detected from 31 out of 282 seed consignments imported from 2006 to 2011. To prevent its introduction to Korea, the seed consignments infested by B. penicillatum were destroyed or reshipped.

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.

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.

Astaxanthin inhibits H2O2-mediated apoptotic cell death in mouse neural progenitor cells via modulation of P38 and MEK signaling pathways.

In the present study, neuroprotective effects of astaxanthin on H2O2-mediated apoptotic cell death using cultured mouse neural progenitor cells (mNPCs) were investigated. To cause apoptotic cell death, mNPCs were pretreated with astaxanthin for 8 h and followed by treatment of 0.3 mM H2O2. Pretreatment of mNPCs with astaxanthin significantly inhibited H2O2-mediated apoptosis and induced cell growth in a dose-dependent manner. In Western blot analysis, astaxanthin-pretreated cells showed the activation of p-Akt, p-MEK, p-ERK, and Bcl-2, and the reduction of p-P38, p-SAPK/JNK, Bax, p-GSK3beta, cytochrome c, caspase-3, and PARP. Because H2O2 triggers caspases activation, this study examined whether astaxanthin can inhibit caspases activation in H2O2-treated mNPCs. After H2O2 treatment, caspases activities were prominently increased but astaxanthin pretreatment significantly inhibited H2O2-mediated caspases activation. Astaxanthin pretreatment also significantly recovered ATP production ability of H2O2-treated cells. These findings indicate that astaxanthin inhibits H2O2-mediated apoptotic features in mNPCs. Inhibition assays with SB203580 (10 microM, a specific inhibitor of p38) and PD98059 (10 microM, a specific inhibitor of MEK) clearly showed that astaxanthin can inhibit H2O2-mediated apoptotic death via modulation of p38 and MEK signaling pathways.

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.

Genome-wide functional analysis of pathogenicity genes in the rice blast fungus.

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.

Cloning and sequence analysis of the beta2-microglobulin transcript from flounder, Paralichthys olivaceous.

Beta2-microglobulin (beta2M) is a protein found free-form in the serum or on the cell surface non-covalently associated with the alpha-chain of the class I major histocompatibility (MHC-I) complex. The full-length cDNA containing beta2M was cloned from flounder, Paralichthys olivaceous. The transcript consists of 1610 nucleotides (nts), including an open reading frame (ORF) of 384 nts encoding a polypeptide of 128 amino acids. The amino acid sequence of beta2M in flounder is 59, 57, 56, and 48% conserved in catfish, rainbow trout, zebrafish, and humans, respectively. Genomic Southern hybridization suggested the presence of a single copy of beta2M in the flounder genome, and reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis detected the beta2M transcript in the head kidney, spleen, body kidney, liver, and muscle tissues of the flounder. PCR amplification and sequence analysis revealed the lack of an intron in the beta2M gene. The phylogenetic analysis confirmed the evolutionary diversion of the beta2M protein among warm-blooded vertebrates and fish, and the separation between freshwater and seawater fish.

Extracellular matrix protein gene, EMP1, is required for appressorium formation and pathogenicity of the rice blast fungus, Magnaporthe grisea.

Magnaporthe grisea, the causal fungus of rice blast, forms a specialized infection structure called an appressorium that is crucial for host plant penetration. A cDNA clone of M. grisea, showing strong sequence homology to FEM1 of Fusarium oxysporum and encoding an extracellular matrix protein, was isolated during an expressed sequence tag (EST) analysis of an appressorium cDNA library and named extracellular matrix protein 1 (EMP1). Sequence analysis of the corresponding genomic clone revealed that EMP1 contains an open reading frame of 685 nucleotides encoding 207 amino acids. The estimated molecular weight of the protein product was 20.5 kDa with a pI of 7.84. It contains an 18 amino acid N-terminal secretion signal sequence, as well as four potential N-glycosylation sites. At its C-terminus, the protein contains a 16 amino acid sequence with the characteristics of a glycosylphosphatidylinositol (GPI) anchor addition signal. Northern blot analysis showed that EMP1 transcripts accumulate during appressorium formation but not during vegetative growth. An EMP1 null mutant, emp1, generated by targeted gene disruption, exhibited reduced levels of appressorium formation and pathogenicity but no effect on mycelial growth rate or conidiation ability. These data suggest that EMP1 plays important roles in appressorium formation and the pathogenicity of M. grisea.

Cloning, sequencing and functional analysis of Magnaporthe grisea MVP1 gene, a hex-1 homolog encoding a putative 'woronin body' protein.

A hex-1 homolog named MVP1 was isolated from an appressoria cDNA library of the rice blast fungus Magnaporthe grisea. The transcript of approximately 1.6 kb contains 546 bp of coding sequence with a 3' untranslated region about 168 bp long and a 5' untranslated region about 870 bp long. Southern gel blot analysis of genomic DNA following digestion with three restriction enzymes (BamHI, EcoRI, HindIII) indicated that the gene exists as a single copy in M. grisea genome. RNA gel blot analyses showed that MVP1 was highly expressed in germinating conidia and the mycelial stage compared to appressoria or non-germinated conidia. MVP1 showed a high degree of homology to the hex-1 gene recently described to encode a major protein in the woronin bodies of Neurospora crassa. Double homologous recombination was used to replace MVP1 with the hyg(R) gene. MVP1 knockout mutants showed apical swellings when grown on agar plates containing 2% sorbose but they were not impaired in any other vegetative or pathogenic properties evaluated. The pathological and other phenotypic consequences of gene disruption are discussed.

Calcium restores prepenetration morphogenesis abolished by polyamines in Colletotrichum gloeosporioides infecting red pepper.

Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, to infect its host, red pepper. Polyamines (putrescine, spermidine, and spermine) as well as S-adenosyl methionine inhibitor, methylglyoxal-bis-guanyl hydrazone (MGBG), impaired conidial germination and appressorium formation of C. gloeosporioides. Curtailment of cell differentiation by polyamines and MGBG was more evident in conidial germination than in appressorium development. Exogenous addition of calcium restored conidial germination and appressorium formation and expression of calmodulin-encoding gene (CgCaM) inhibited by polyamines. Taken together, proper regulation of intracellular polyamine concentration is indispensable for conidial germination and appressorium formation, and involved in Ca(2+)/calmodulin-dependent signaling pathways of C. gloeosporioides infecting red pepper.

Isolation of a novel antimicrobial peptide gene (Sp-AMP) homologue from Pinus sylvestris (Scots pine) following infection with the root rot fungus Heterobasidion annosum.

A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional members of the Sp-AMP family (Sp-AMPs 2-4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia (MiAMP1), which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum-conifer pathosystem is discussed.

Large scale parallel analysis of gene expression during infection-related morphogenesis of Magnaporthe grisea.

SUMMARY The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice. To initiate the infection of host tissues, conidia elaborate germ tubes that differentiate specialized infection structures called appressoria. Microarrays composed of 3500 cDNAs of M. grisea were prepared for the identification of genes that are specifically up- or down-regulated during appressorium formation. Gene expression in ungerminated conidia, during appressorium formation, and during mycelial growth was investigated with a novel highly sensitive dendrimer based detection system. Transcripts of 85 different genes were found to be more abundant in ungerminated conidia and/or in conidia with developing appressoria than in vegetative mycelia. Nineteen of these showed higher expression in both ungerminated conidia and developing appressoria than in mycelia, suggesting that their expression remains elevated during the early stage of fungal infection. The expression of 18 genes was higher in ungerminated conidia than in developing appressoria, indicating their possible role in the germination process or maintaining dormancy. Transcripts of 47 genes were found to be more abundant in developing appressoria than in ungerminated conidia, suggesting that their expression is induced during appressorium formation. Several of these genes, including a chitin binding protein and infection structure specific protein MIF23, were previously shown to be preferentially expressed during appressorium formation. However, the expression of many of these genes has not been reported prior to this analysis. In contrast, transcripts of 38 different genes were found to be more abundant in mycelia than in developing appressoria. A Northern blot analysis of selected genes was consistent with the microarray results. Results from this study provide a powerful resource for furthering our understanding of gene expression during infection-related morphogenesis and for the functional analysis of M. grisea genes involved in fungal infection.

Two novel fungal virulence genes specifically expressed in appressoria of the rice blast fungus.

The PMK1 mitogen-activated protein kinase gene regulates appressorium formation and infectious hyphae growth in the rice blast fungus. To further characterize this mitogen-activated protein kinase pathway, we constructed a subtraction library enriched for genes regulated by PMK1. Two genes identified in this library, GAS1 and GAS2, encode small proteins that are homologous with gEgh16 of the powdery mildew fungus. Both were expressed specifically during appressorium formation in the wild-type strains, but neither was expressed in the pmk1 mutant. Mutants deleted in GAS1 and GAS2 had no defect in vegetative growth, conidiation, or appressoria formation, but they were reduced in appressorial penetration and lesion development. Interestingly, deletion of both GAS1 and GAS2 did not have an additive effect on appressorial penetration and lesion formation. The GAS1-green fluorescent protein and GAS2-green fluorescent protein fusion proteins were expressed only in appressoria and localized in the cytoplasm. These two genes may belong to a class of proteins specific for filamentous fungi and function as novel virulence factors in fungal pathogens.

Cloning of disease-resistance homologues in end sequences of BAC clones linked to Fom-2, a gene conferring resistance to Fusarium wilt in melon (Cucumis melo L.).

Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and I of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.