Sook Young Park

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

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.

A Quick and Safe Method for Fungal DNA Extraction

DNA-based studies, including cloning and genotyping, have become routine in fungal research laboratories. However, preparation of high-quality DNA from fungal tissue requires much time and labor and is often a limiting step for high-throughput experiments. We have developed a quick and safe (QS) DNA extraction method for fungi. Time efficiency and safety in the QS method were achieved by using plate-grown mycelia as the starting material, by eliminating phenol-chloroform extraction procedures, and by deploying a simple electric grinder. This QS method is applicable not only to a broad range of microbial eukaryotes, including true fungi and oomycetes, but also to lichens and plants.

Global Expression Profiling of Transcription Factor Genes Provides New Insights into Pathogenicity and Stress Responses in the Rice Blast Fungus

Because most efforts to understand the molecular mechanisms underpinning fungal pathogenicity have focused on studying the function and role of individual genes, relatively little is known about how transcriptional machineries globally regulate and coordinate the expression of a large group of genes involved in pathogenesis. Using quantitative real-time PCR, we analyzed the expression patterns of 206 transcription factor (TF) genes in the rice blast fungus Magnaporthe oryzae under 32 conditions, including multiple infection-related developmental stages and various abiotic stresses. The resulting data, which are publicly available via an online platform, provided new insights into how these TFs are regulated and potentially work together to control cellular responses to a diverse array of stimuli. High degrees of differential TF expression were observed under the conditions tested. More than 50% of the 206 TF genes were up-regulated during conidiation and/or in conidia. Mutations in ten conidiation-specific TF genes caused defects in conidiation. Expression patterns in planta were similar to those under oxidative stress conditions. Mutants of in planta inducible genes not only exhibited sensitive to oxidative stress but also failed to infect rice. These experimental validations clearly demonstrated the value of TF expression patterns in predicting the function of individual TF genes. The regulatory network of TF genes revealed by this study provides a solid foundation for elucidating how M. oryzae regulates its pathogenesis, development, and stress responses.

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10‐mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p < 0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p < 0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p < 0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury. Copyright

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells

Summary 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 adhesion-dependent 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 cells 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 adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer.

Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea.

A previous study of the diversity and population structure of the rice blast fungus, Magnaporthe oryzae, over a 20-year period in Korea, found novel fingerprint haplotypes each year, and the authors hypothesized that populations might experience annual bottlenecks. Based on this model, we predicted that M. oryzae populations would have little or no genetic differentiation among geographic regions because rice blast is commonly found throughout Korea each year and M. oryzae would have to disperse from small populations surviving annually between rice crops. To test this hypothesis, we sampled M. oryzae from rice fields in eight provinces in Korea in a single year (1999). In four provinces, we sampled from a set of rice cultivars commonly grown in commercial fields (group I); because of low disease incidence in four other provinces, we could not sample from commercial fields and instead sampled from scouting plots of different cultivars set up for detecting new pathotypes of M. oryzae (group II). All isolates were genotyped with DNA fingerprint probes MGR586 and MAGGY, a telomere-linked gene family member TLH1, the PWL2 host specificity gene and mating type. Fingerprint haplotypes clustered into two distinct lineages corresponding to the two sets of cultivars (groups I and II), with haplotype similarities of 71% between lineages and >76% within lineages. Isolates from the same cultivar within group I were genetically differentiated among locations, and isolates within the same location were differentiated among cultivars. Differentiation for TLH1 and PWL2 was significant (P < 0.03), but not as strong as for fingerprint markers. Similar analyses were not possible among group II isolates because too few isolates were available from any one cultivar. All isolates were in the same mating type, Mat1-1, ruling out sexual reproduction as a source of novel haplotypes. When the 1999 samples were compared with the historical samples from the previous study, haplotypes of group I formed a separate cluster, while those of group II clustered with haplotypes from the historical sample. Altogether, geographic subdivision, monomorphism of mating type, and correlation of haplotypes to sets of cultivars are not consistent with the hypothesis of repeated turnover of haplotypes. Instead, the previous correlations of haplotypes to year might have been caused by inadequate sampling of haplotypes each year, highlighting the need for studies of population genetics to be conducted with systematic samples collected to address specific questions.

3D braid scaffolds for regeneration of articular cartilage.

Regenerating articular cartilage in vivo from cultured chondrocytes requires that the cells be cultured and implanted within a biocompatible, biodegradable scaffold. Such scaffolds must be mechanically stable; otherwise chondrocytes would not be supported and patients would experience severe pain. Here we report a new 3D braid scaffold that matches the anisotropic (gradient) mechanical properties of natural articular cartilage and is permissive to cell cultivation. To design an optimal structure, the scaffold unit cell was mathematically modeled and imported into finite element analysis. Based on this analysis, a 3D braid structure with gradient axial yarn distribution was designed and manufactured using a custom-built braiding machine. The mechanical properties of the 3D braid scaffold were evaluated and compared with simulated results, demonstrating that a multi-scale approach consisting of unit cell modeling and continuum analysis facilitates design of scaffolds that meet the requirements for mechanical compatibility with tissues.

Surface coating of hydroxyapatite on silk nanofiber through biomineralization using ten times concentrated simulated body fluid and the evaluation for bone regeneration

AbstractThe biomineralization of hydroxyapatite (HA) on silk nanofiber surface was explored to create organic/inorganic composite scaffold for bone tissue engineering. The porous silk nanofibers matrix was prepared by electrospinning using methanol coagulation bath as a collector and the deposition and coating of HA crystals were proceeded through biomineralization process by immersing the nanofibers in ten times concentrated simulated body fluid (10X SBF). It was found that uniform surface coating of silk nanofibers with HA crystals can be easily obtained by using 10X SBF within short immersion time (60–90 min in this study). The morphological structure of the Silk/HA composite nanofiber was characterized by field emission scanning electron microscopy (FE-SEM) images and structural evidence of HA deposition onto the silk nanofibers were also performed by energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Methylthiazol tetrazolium (MTT) assay confirmed that the cell adhesion and proliferation of osteoblast cells on this composite scaffold was excellent and micro computed tomography (micro-CT) image also showed that better ability of bone regeneration occurred for the Silk/HA composite nanofiber scaffold after 8 weeks implantation. As a result of biological evaluation, the HA coated silk nanofiber scaffold can have a high potential for using in bone tissue regeneration.

Phytophthora Database 2.0: Update and Future Direction

The online community resource Phytophthora database (PD) was developed to support accurate and rapid identification of Phytophthora and to help characterize and catalog the diversity and evolutionary relationships within the genus. Since its release in 2008, the sequence database has grown to cover 1 to 12 loci for ≈2,600 isolates (representing 138 described and provisional species). Sequences of multiple mitochondrial loci were added to complement nuclear loci-based phylogenetic analyses and diagnostic tool development. Key characteristics of most newly described and provisional species have been summarized. Other additions to improve the PD functionality include: (i) geographic information system tools that enable users to visualize the geographic origins of chosen isolates on a global-scale map, (ii) a tool for comparing genetic similarity between isolates via microsatellite markers to support population genetic studies, (iii) a comprehensive review of molecular diagnostics tools and relevant references, (iv) sequence alignments used to develop polymerase chain reaction-based diagnostics tools to support their utilization and new diagnostic tool development, and (v) an online community forum for sharing and preserving experience and knowledge accumulated in the global Phytophthora community. Here we present how these improvements can support users and discuss the PDs future direction.