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Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus Magnaporthe oryzae, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neofunctionalization of some genes during infection. Comprehensive profiling of the pattern of genomewide AS during multiple stages of rice-M. oryzae interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection. [ABSTRACT FROM AUTHOR]

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica. [ABSTRACT FROM AUTHOR]

Lichens are a natural source of bioactive compounds. Cladonia metacorallifera var. reagens KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, thamnolic, and didymic acids. However, these metabolites have not been detected in isolated mycobionts. We investigated the effects of six carbon sources on metabolite biosynthesis in the C. metacorallifera mycobiont. Red pigments appeared only in Lilly and Barnett's media with fructose at 15 °C after 3 weeks of culture and decreased after 6 weeks. We purified these red pigments using preparativescale high performance liquid chromatography and analyzed them via nuclear magnetic resonance. Results indicated that 1% fructose-induced cristazarin and 6-methylcristazarin production under light conditions. In total, 27 out of 30 putative polyketide synthase genes were differentially expressed after 3 weeks of culture, implying that these genes may be required for cristazarin production in C. metacorallifera. Moreover, the white collar genes Cmwc-1 and Cmwc-2 were highly upregulated at all times under light conditions, indicating a possible correlation between cristazarin production and gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC50 values of 18.2, 26.1, and 30.9 μg/mL, respectively, which highlights the value of cristazarin. Overall, our results suggest that 1% fructose under light conditions is required for cristazarin production by C. metacorallifera mycobionts, and cristazarin could be a good bioactive compound. [ABSTRACT FROM AUTHOR]

Scab disease caused by Venturia nashicola is of agroeconomic importance in cultivation of Asian pear. However, little is known about the degree of genetic diversity in the populations of this pathogen. In this study, we collected 55 isolates from pear scab lesions in 13 major cultivation areas in Korea and examined the diversity using sequences of internal transcribed spacer (ITS) region, b-tubulin (TUB2), and translation elongation factor-1a (TEF-1a) genes as molecular markers. Despite a low level of overall sequence variation, we found three distinctive subgroups from phylogenetic analysis of combined ITS, TUB2, and TEF-1a sequences. Among the three subgroups, subgroup 1 (60% of isolates collected) was predominant compared to subgroup 2 (23.6%) or subgroup 3 (16.4%) and was distributed throughout Korea. To understand the genetic diversity among the subgroups, RAPD analysis was performed. The isolates yielded highly diverse amplicon patterns and none of the defined subgroups within the dendrogram were supported by bootstrap values greater than 30%. Moreover, there is no significant correlation between the geographical distribution and the subgroups defined by molecular phylogeny. Our data suggest a low level of genetic diversification among the populations of V. nashicola in Korea. [ABSTRACT FROM AUTHOR]

Lichen genus Stereocaulon (Schreb.) Hoffm is distributed throughout the world. Although 15 Stereocaulon species have been recorded in Korea, no detailed taxonomic or revisionary research has been conducted for nearly two decades. In this study, we collected 260 putative Stereocaulon spp. samples and identified the species based on morphological, chemical, and molecular characteristics. From the collected samples, 10 species of Stereocaulon were identified, nine of which had already been reported, although this was the first report for the tenth, 5. octomerellum Hue, in Korea. General characteristics of Stereocaulon spp. include coralloid phyllocladia and tubercular cephalodia; however, the specimen first collected in Korea was a rare species with tomentum on the pseudopodetia. The specimen of 5. octomerellum is characterized by the presence of a primary thallus, granule to short coralloid phyllocladia, and pseudopodetia up to 1 cm in size, with tubercular cephalodia. To determine the phylogeny of the specimens, we compared the ITS sequences of ribosomal DNA and the (3-tubulin gene sequences. Phylogenetic analysis showed that the Korean Stereocaulon species were monophyletic and placed in the previous phylogenetic classification. Species of S. intermedium and S. exutum, however, were polyphyletic, and are morphologically variable and widespread species. Overall, we present here detailed morphological and chemical descriptions of each species identified and a revised key of all known Stereocaulon species in South Korea. [ABSTRACT FROM AUTHOR]

A total of 121 species of lichens belonging to the genus Arthothelium have been described to date, most of which have been found in tropical regions. Here, we describe the discovery of a novel Arthothelium species for the first time in South Korea. Until now, Arthothelium ruanum was the only Arthothelium species reported in South Korea. Among the 113 specimens collected in this study, we identified A. ruanum and a putative new species, Arthothelium punctatum (J. S. Park & J.-S. Hur, sp. nov.). The diagnostic characters of A. punctatum are as follows: apothecia punctate, shortly elongate to branched, small, 0.1-0.2 mm wide, hypothecium hyaline to pale brown and obovate to broadly ellipsoid, muriform ascospores, 29.5-44.6 × 12.2-18.2 µm. The new species was found in Mt. Seokbyeong at an altitude of 790 m on smooth bark. Upon phylogenic analysis, the putative new species, A. punctatum, was separated from other Arthothelium species although the specimens analyzed were clustered with Arthoniaceae in phylogenetic trees based on both the mitochondrial small subunit (mtSSU) sequence and combined mtSSU and nuclear ribosomal large subunit sequences. Our data clearly indicate that this species is a new species belonging to the family Arthoniaceae. To elucidate the taxonomic characteristics of the new species, we provide morphological descriptions and a distribution map. [ABSTRACT FROM AUTHOR]

The crustose lichen genus Pertusaria comprises over ca. 800 species worldwide. In total, 20 Pertusaria species were localized to the Mt. Sorak and Jeju-do in Korea. To date, information regarding the distribution of Pertusaria species in the South Korean peninsula is scarce. In this study, we collected Pertusaria species across South Korea and identified them based on morphological, chemical, and molecular characteristics. Of the 387 samples collected, we identified 24 taxa and 1 variety, of which 17 were previously recorded, and 6 taxa were newly found in South Korea (P. leioplaca, P. leucosora var. violascens, P. texana, P. thiospoda, P. thwaitesii, and P. xanthodes), 2 known species were transferred to Varicellaria (Varicellaria lactea and V. velata), one species was transferred to Variolaria as a new record (Variolaria multipunctoides) and one was a new species (P. jogyeensis J. S. Park & J.-S. Hur, sp. nov.). Characteristics of the newly discovered species, P. jogyeensis, are as follows: smooth to bumpy thallus, scattered to crowded poriform apothecia, blackish ostioles, definitely sunken, thin yellowish green rims around ostioles, 8-spored ascus, and the presence of perlatolic acid and thiophaninic acid (chlorinated xanthone). Phylogenetic studies on P. jogyeensis based on the mitochondrial small subunit sequence revealed proximity to P. flavicans and P. texana, and supported its classification as a new species within the genus Pertusaria. Additionally, we describe the chemical composition and morphology of all listed species in detail and provide an artificial key for identification. [ABSTRACT FROM AUTHOR]

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples. [ABSTRACT FROM AUTHOR]

Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesiondependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesiondependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer. [ABSTRACT FROM AUTHOR]

In eukaryotes, microbodies called peroxisomes play important roles in cellular activities during the life cycle. Previous studies indicate that peroxisomal functions are important for plant infection in many phytopathogenic fungi, but detailed relationships between fungal pathogenicity and peroxisomal function still remain unclear. Here we report the importance of peroxisomal protein import through PTS2 (Peroxisomal Targeting Signal 2) in fungal development and pathogenicity of Magnaporthe oryzae. Using an Agrobacterium tumefaciens-mediated transformation library, a pathogenicity-defective mutant was isolated from M. oryzae and identified as a T-DNA insert in the PTS2 receptor gene, MoPEX7. Gene disruption of MoPEX7 abolished peroxisomal localization of a thiolase (MoTHL1) containing PTS2, supporting its role in the peroxisomal protein import machinery. ΔMopex7 showed significantly reduced mycelial growth on media containing short-chain fatty acids as a sole carbon source. ΔMopex7 produced fewer conidiophores and conidia, but conidial germination was normal. Conidia of ΔMopex7 were able to develop appressoria, but failed to cause disease in plant cells, except after wound inoculation. Appressoria formed by ΔMopex7 showed a defect in turgor generation due to a delay in lipid degradation and increased cell wall porosity during maturation. Taken together, our results suggest that the MoPEX7-mediated peroxisomal matrix protein import system is required for fungal development and pathogenicity M. oryzae. [ABSTRACT FROM AUTHOR]

The three dimensional (3-D) poly(trimethylenecarbonate- co- ε-caprolactone)-block-poly( p-dioxanone) scaffold was made using a wet electrospinning method and its application as a tissue engineered matrix was evaluated for bone regeneration. The scaffold was highly porous (90%) and interconnected among pores. Under scanning electron microscopy, the cells of the center of the scaffold showed healthy well attached shape even at 4 days after seeding. The osteoblastic MC3T3-E1 cells proliferated 1.2 times faster at 4 day, 1.5 times faster at 7 days after seeding as compared with the control in the scaffold ( P < 0.05). The activity of alkaline phosphatase, a bone formation marker, of cells seeded in the scaffold was nearly four times faster compared to control 28 days after seeding ( P < 0.05). Taken together, newly developed 3-D poly(trimethylenecarbonate- co- ε-caprolactone)-block-poly( p-dioxanone) scaffold is a promising candidate for bone regeneration. [ABSTRACT FROM AUTHOR]

Background: Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction. Results: A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 fulllength fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence. Conclusion: This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis. [ABSTRACT FROM AUTHOR]

The appropriate development of conidia and appressoria is critical in the disease cycle of many fungal pathogens, including Magnaporthe oryzae. A total of eight genes (MoHOX1 to MoHOX8) encoding putative homeobox transcription factors (TFs) were identified from the M. oryzae genome. Knockout mutants for each MoHOX gene were obtained via homologydependent gene replacement. Two mutants, ΔMohox3 and ΔMohox5, exhibited no difference to wild-type in growth, conidiation, conidium size, conidial germination, appressorium formation, and pathogenicity. However, the ΔMohox1 showed a dramatic reduction in hyphal growth and increase in melanin pigmentation, compared to those in wild-type. ΔMohox4 and ΔMohox6 showed significantly reduced conidium size and hyphal growth, respectively. ΔMohox8 formed normal appressoria, but failed in pathogenicity, probably due to defects in the development of penetration peg and invasive growth. It is most notable that asexual reproduction was completely abolished in ΔMohox2, in which no conidia formed. ΔMohox2 was still pathogenic through hypha-driven appressoria in a manner similar to that of the wild-type. However, ΔMohox7 was unable to form appressoria either on conidial germ tubes, or at hyphal tips, being non-pathogenic. These factors indicate that M. oryzae is able to cause foliar disease via hyphal appressorium-mediated penetration, and MoHOX7 is mutually required to drive appressorium formation from hyphae and germ tubes. Transcriptional analyses suggest that the functioning of M. oryzae homeobox TFs is mediated through the regulation of gene expression and is affected by cAMP and Ca2+ signaling and/or MAPK pathways. The divergent roles of this gene set may help reveal how the genome and regulatory pathways evolved within the rice blast pathogen and close relatives. [ABSTRACT FROM AUTHOR]

For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant. We demonstrate that a novel pathogenicity gene, DES1, in Magnaporthe oryzae regulates counter-defenses against host basal resistance. The DES1 gene was identified by screening for pathogenicity-defective mutants in a T-DNA insertional mutant library. Bioinformatic analysis revealed that this gene encodes a serine-rich protein that has unknown biochemical properties, and its homologs are strictly conserved in filamentous Ascomycetes. Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration. Conidial size of the mutant became smaller than that of the wild type, but the mutant displayed no defects on cell wall integrity. The Δdes1 mutant was hypersensitive to exogenous oxidative stress and the activity and transcription level of extracellular enzymes including peroxidases and laccases were severely decreased in the mutant. In addition, ferrous ion leakage was observed in the Δdes1 mutant. In the interaction with a susceptible rice cultivar, rice cells inoculated with the Ddes1 mutant exhibited strong defense responses accompanied by brown granules in primary infected cells, the accumulation of reactive oxygen species (ROS), the generation of autofluorescent materials, and PR gene induction in neighboring tissues. The Δdes1 mutant displayed a significant reduction in infectious hyphal extension, which caused a decrease in pathogenicity. Notably, the suppression of ROS generation by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, resulted in a significant reduction in the defense responses in plant tissues challenged with the Δdes1 mutant. Furthermore, the Δdes1 mutant recovered its normal infectious growth in DPI-treated plant tissues. These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense. [ABSTRACT FROM AUTHOR]

In planta secretion of fungal pathogen proteins, including effectors destined for the plant cell cytoplasm, is critical for disease progression. However, little is known about the endoplasmic reticulum (ER) secretion mechanisms used by these pathogens. To determine if normal ER function is crucial for fungal pathogenicity, Magnaporthe oryzae genes encoding proteins homologous to yeast Lhs1 p and Kar2p, members of the heat shock protein 70 family in Saccharomyces cerevisiae, were cloned and characterized. Like their yeast counterparts, both LHS1 and KAR2 proteins localized in the ER and functioned in an unfolded protein response (UPR) similar to the yeast UPR. Mutants produced by disruption of LHS1 were viable but showed a defect in the translocation of proteins across the ER membrane and reduced activities of extracellular enzymes. The Δlhs1 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice (Oryza sativa) plants. This mutant also had defects in the induction of the Pi-ta resistance gene--mediated hypersensitive response and in the accumulation of fluorescently-labeled secreted effector proteins in biotrophic interfacial complexes. Our results suggest that proper processing of secreted proteins, including effectors, by chaperones in the ER is requisite for successful disease development and for determining host-pathogen compatibility via the gene-for-gene interaction. [ABSTRACT FROM AUTHOR]

We made a three-dimensional (3-D) nanofibrous fibroin scaffold (NFS) with high porosity (94%) and examined its feasibility in bone regeneration. Under scanning electron microscopy, MC3T3-E1 preosteoblasts on the scaffold showed more spread on the first day after seeding compared with a 2-D scaffold. MTT assay showed significantly increased proliferation in 3-D NFS compared with 2-D NFS 7 days after seeding ( P < 0.05). Western immunoblotting for activated paxillin, FAK, AKT, C-Src, and ERK1/2 antibodies showed signals from the extracellular matrix were significantly increased in 3-D NFS. Newly developed 3-D electrospun NFS may be a good candidate for use in bone regeneration. [ABSTRACT FROM AUTHOR]

grobacterium umefaciens- ediated ransformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T-DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant-pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T-DNA integration events in fungi and the development of an informatics infrastructure, termed a -DNA nalysis latform (TAP). We identified a total of 1110 -DNA- agged ocations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T-DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T-DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T-DNA borders and flanking genomic DNA sequences revealed that T-DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis. [ABSTRACT FROM AUTHOR]

A correction to the article "Genome-Wide Analysis of T-DNA Integration Into the Chromosomes of Magnaporthe oryzae" that was published in a 2007 issue is presented.