Bongsoo Park

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.

Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

ABSTRACT Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolates clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org ) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium ). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

Fungal cytochrome P450 database.

BackgroundCytochrome P450 enzymes play critical roles in fungal biology and ecology. To support studies on the roles and evolution of cytochrome P450 enzymes in fungi based on rapidly accumulating genome sequences from diverse fungal species, an efficient bioinformatics platform specialized for this super family of proteins is highly desirable.ResultsThe Fungal Cytochrome P450 Database (FCPD) archives genes encoding P450s in the genomes of 66 fungal and 4 oomycete species (4,538 in total) and supports analyses of their sequences, chromosomal distribution pattern, and evolutionary histories and relationships. The archived P450s were classified into 16 classes based on InterPro terms and clustered into 141 groups using tribe-MCL. The proportion of P450s in the total proteome and class distribution in individual species exhibited certain taxon-specific characteristics.ConclusionThe FCPD will facilitate systematic identification and multifaceted analyses of P450s at multiple taxon levels via the web. All data and functions are available at the web site http://p450.riceblast.snu.ac.kr/.

CFGP: a web-based, comparative fungal genomics platform

Since the completion of the Saccharomyces cerevisiae genome sequencing project in 1996, the genomes of over 80 fungal species have been sequenced or are currently being sequenced. Resulting data provide opportunities for studying and comparing fungal biology and evolution at the genome level. To support such studies, the Comparative Fungal Genomics Platform (CFGP; http://cfgp.snu.ac.kr), a web-based multifunctional informatics workbench, was developed. The CFGP comprises three layers, including the basal layer, middleware and the user interface. The data warehouse in the basal layer contains standardized genome sequences of 65 fungal species. The middleware processes queries via six analysis tools, including BLAST, ClustalW, InterProScan, SignalP 3.0, PSORT II and a newly developed tool named BLASTMatrix. The BLASTMatrix permits the identification and visualization of genes homologous to a query across multiple species. The Data-driven User Interface (DUI) of the CFGP was built on a new concept of pre-collecting data and post-executing analysis instead of the ‘fill-in-the-form-and-press-SUBMIT’ user interfaces utilized by most bioinformatics sites. A tool termed Favorite, which supports the management of encapsulated sequence data and provides a personalized data repository to users, is another novel feature in the DUI.

Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes

BackgroundCytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable “CYPomes” ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme.ResultsThe new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelson’s P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database.ConclusionsFCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families) encoded by five oomycete species. In comparison to the first version, it offers a more comprehensive clan classification, is fully compatible with Nelson’s P450 databases, and has expanded functional categorization. These features will facilitate functional annotation and classification of CYPs encoded by newly sequenced fungal and oomycete genomes. Additionally, the classification system will aid in studying the roles of CYPs in the evolution of fungal adaptation to specific ecological niches.

Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller–nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the FUSARIUM-ID and Fusarium MLST online databases and analysis packages. Analyses of a 190-taxon, two-locus dataset, which included 159 isolates from insects, indicated that: (i) insect-associated fusaria were nested within 10 species complexes spanning the phylogenetic breadth of Fusarium, (ii) novel, putatively unnamed insecticolous species were nested within 8/10 species complexes and (iii) Latin binomials could be applied with confidence to only 18/58 phylogenetically distinct fusaria associated with pest insects. Phylogenetic analyses of an 82-taxon, three-locus dataset nearly fully resolved evolutionary relationships among the 10 clades containing insecticolous fusaria. Multilocus typing of isolates within four species complexes identified surprisingly high genetic diversity in that 63/65 of the fusaria typed represented newly discovered haplotypes. The DNA sequence data, together with corrected ABI sequence chromatograms and alignments, have been uploaded to the following websites dedicated to identifying fusaria: FUSARIUM-ID (http://isolate.fusariumdb.org) at Pennsylvania State University’s Department of Plant Pathology and Fusarium MLST (http://www.cbs.knaw.nl/fusarium) at the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center.

The Promise and Pitfalls of Sequence-Based Identification of Plant-Pathogenic Fungi and Oomycetes

Sequences of selected marker loci have been widely used for the identification of specific pathogens and the development of sequence-based diagnostic methods. Although such approaches offer several advantages over traditional culture-based methods for pathogen diagnosis and identification, they have their own pitfalls. These include erroneous and incomplete data in reference databases, poor or oversimplified interpretation of search results, and problems associated with defining species boundaries. In this letter, we outline the potential benefits and drawbacks of using sequence data for identification and taxonomic deduction of plant-pathogenic fungi and oomycetes, using phytophthora as a primary example. We also discuss potential remedies for these pitfalls and address why coordinated community efforts are essential to make such remedies more efficient and robust.

Phytophthora Database: A Forensic Database Supporting the Identification and Monitoring of Phytophthora

Phytophthora spp. represent a serious threat to agricultural and ecological systems. Many novel Phytophthora spp. have been reported in recent years, which is indicative of our limited understanding of the ecology and diversity of Phytophthora spp. in nature. Systematic cataloging of genotypic and phenotypic information on isolates of previously described species serves as a baseline for identification, classification, and risk assessment of new Phytophthora isolates. The Phytophthora Database (PD) was established to catalog such data in a web-accessible and searchable format. To support the identification of new Phytophthora isolates via comparison of their sequences at one or more loci with the corresponding sequences derived from the isolates archived in the PD, we generated and deposited sequence data from more than 1,500 isolates representing the known diversity in the genus. Data search and analysis tools in the PD include BLAST, Phyloviewer (a program for building phylogenetic trees using sequences of selected isolates), and Virtual Gel (a program for generating expected restriction patterns for given sequences). The PD also provides a customized means of storing and sharing data via the web. The PD serves as a model that easily can be adopted to develop databases for other important pathogen groups.

A Comprehensive and High-Resolution Genome-wide Response of p53 to Stress

Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.