Biomedical subjects
Ralph A Dean
Publications and source records attributed to Ralph A Dean.
The role of transposable element clusters in genome evolution and loss of synteny in the rice blast fungus Magnaporthe oryzae.
BACKGROUND: Transposable elements are abundant in the genomes of many filamentous fungi, and have been implicated as major contributors to genome rearrangements and as sources of genetic variation. Analyses of fungal genomes have also revealed that transposable elements are largely confined to distinct clusters within the genome. Their impact on fungal genome evolution is not well understood. Using the recently available genome sequence of the plant pathogenic fungus Magnaporthe oryzae, combined with additional bacterial artificial chromosome clone sequences, we performed a detailed analysis of the distribution of transposable elements, syntenic blocks, and other features of chromosome 7. RESULTS: We found significant levels of conserved synteny between chromosome 7 and the genomes of other filamentous fungi, despite more than 200 million years of divergent evolution. Transposable elements are largely restricted to three clusters located in chromosomal segments that lack conserved synteny. In contradiction to popular evolutionary models and observations from other model organism genomes, we found a positive correlation between recombination rate and the distribution of transposable element clusters on chromosome 7. In addition, the transposable element clusters are marked by more frequent gene duplications, and genes within the clusters have greater sequence diversity to orthologous genes from other fungi. CONCLUSION: Together, these data suggest that transposable elements have a profound impact on the M. oryzae genome by creating localized segments with increased rates of chromosomal rearrangements, gene duplications and gene evolution.
Alkahest NuclearBLAST : a user-friendly BLAST management and analysis system.
BACKGROUND: Sequencing of EST and BAC end datasets is no longer limited to large research groups. Drops in per-base pricing have made high throughput sequencing accessible to individual investigators. However, there are few options available which provide a free and user-friendly solution to the BLAST result storage and data mining needs of biologists. RESULTS: Here we describe NuclearBLAST, a batch BLAST analysis, storage and management system designed for the biologist. It is a wrapper for NCBI BLAST which provides a user-friendly web interface which includes a request wizard and the ability to view and mine the results. All BLAST results are stored in a MySQL database which allows for more advanced data-mining through supplied command-line utilities or direct database access. NuclearBLAST can be installed on a single machine or clustered amongst a number of machines to improve analysis throughput. NuclearBLAST provides a platform which eases data-mining of multiple BLAST results. With the supplied scripts, the program can export data into a spreadsheet-friendly format, automatically assign Gene Ontology terms to sequences and provide bi-directional best hits between two datasets. Users with SQL experience can use the database to ask even more complex questions and extract any subset of data they require. CONCLUSION: This tool provides a user-friendly interface for requesting, viewing and mining of BLAST results which makes the management and data-mining of large sets of BLAST analyses tractable to biologists.
The genome sequence of the rice blast fungus Magnaporthe grisea.
Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.
'PACLIMS': a component LIM system for high-throughput functional genomic analysis.
BACKGROUND: Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the approximately 11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. RESULTS: The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. CONCLUSION: Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.
Novel G-protein-coupled receptor-like proteins in the plant pathogenic fungus Magnaporthe grisea.
BACKGROUND: The G-protein-coupled receptors (GPCRs) are one of the largest protein families in human and other animal genomes, but no more than 10 GPCRs have been characterized in fungi. Do fungi contain only this handful or are there more receptors to be discovered? We asked this question using the recently sequenced genome of the fungal plant pathogen Magnaporthe grisea. RESULTS: Proteins with significant similarity to fungus-specific and other eukaryotic GPCRs were identified in M. grisea. These included homologs of known fungal GPCRs, the cAMP receptors from Dictyostelium, and a steroid receptor mPR. We also identified a novel class of receptors typified by PTH11, a cell-surface integral membrane protein required for pathogenicity. PTH11 has seven transmembrane regions and an amino-terminal extracellular cysteine-rich EGF-like domain (CFEM domain), a characteristic also seen in human GPCRs. Sixty-one PTH11-related proteins were identified in M. grisea that shared a common domain with homologs in Neurospora crassa and other fungi belonging to this subphylum of the Ascomycota (the Pezizomycotina). None was detected in other fungal groups (Basidiomycota or other Ascomycota subphyla, including yeasts) or any other eukaryote. The subclass of PTH11 containing the CFEM domain is highly represented in M. grisea. CONCLUSION: In M. grisea we identified homologs of known GPCRs and a novel class of GPCR-like receptors specific to filamentous ascomycetes. A member of this new class, PTH11, is required for pathogenesis, thus suggesting roles in pathogenicity for other members. The identified classes constitute the largest number of GPCR-like proteins reported in fungi to date.
Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction.
To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.
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.
BAC end sequences and a physical map reveal transposable element content and clustering patterns in the genome of Magnaporthe grisea.
Transposable elements (TEs) are viewed as major contributors to the evolution of fungal genomes. Genomic resources such as BAC libraries are an underutilized resource for studying genome-wide TE distribution. Using the BAC end sequences and physical map that are available for the rice blast fungus, Magnaporthe grisea, we describe a likelihood ratio test designed to identify clustering of TEs in the genome. A significant variation in the distribution of three TEs, MAGGY, MGL, and Pot2 was observed among the fingerprint contigs of the physical map. We utilized a draft sequence of M. grisea chromosome 7 to validate our results and found a similar pattern of clustering. By examining individual BAC end sequences, we found evidence for 11 unique integrations of MAGGY or MGL into Pot2 but no evidence for the reciprocal integration of Pot2 into another TE. This suggests that: (a) the presence of Pot2 in the genome predates that of the other TEs, (b) Pot2 was less transpositionally active than other TEs, or (c) that MAGGY and MGL have integration site preference for Pot2. High transition/transversion mutation ratios as well as bias in transition site context was observed in MAGGY and MGL elements, but not in Pot2 elements. These features are consistent with the effects of a Repeat-Induced Point (RIP) mutation-like process occurring in MAGGY and MGL elements. This study illustrates the general utility of a physical map and BAC end sequences for the study of genome-wide repetitive DNA content and organization.
Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization.
Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.
Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis.
Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508-512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are similar, a detailed protocol for LongSAGE library construction has not been reported yet, and several technical difficulties associated with concatemer cloning and purification have not been solved. In this study, we report a substantially improved LongSAGE method called Robust-LongSAGE, which has four major improvements when compared with the previously reported protocols. First, a small amount of mRNA (50 ng) was enough for a library construction. Second, enhancement of cDNA adapter and ditag formation was achieved through an extended ligation period (overnight). Third, only 20 ditag polymerase chain reactions were needed to obtain a complete library (up to 90% reduction compared with the original protocols). Fourth, concatemers were partially digested with NlaIII before cloning into vector (pZEro-1), greatly improving cloning efficiency. The significant contribution of Robust-LongSAGE is that it solved the major technical difficulties, such as low cloning efficiency and small insert sizes associated with existing SAGE and LongSAGE protocols. Using this protocol, one can generate two to three libraries, each containing over 4.5 million tags, within a month. We recently have constructed five libraries from rice (Oryza sativa), one from maize (Zea mays), and one from the rice blast fungus (Magnaporthe grisea).
The Fusarium wilt resistance locus Fom-2 of melon contains a single resistance gene with complex features.
The soil-borne fungus Fusarium oxysporum f.sp. melonis causes significant losses in the cultivated melon, a key member of the economically important family, the Cucurbitaceae. Here, we report the map-based cloning and characterization of the resistance gene Fom-2 that confers resistance to race 0 and 1 of this plant pathogen. Two recombination events, 75 kb apart, were found to bracket Fom-2 after screening approximately 1324 gametes with PCR-based markers. Sequence analysis of the Fom-2 interval revealed the presence of two candidate genes. One candidate gene showed significant similarity to previously characterized resistance genes. Sequence analysis of this gene revealed clear polymorphisms between resistant and susceptible materials and was therefore designated as Fom-2. Analysis of susceptible breeding lines (BL) presenting a haplotype very similar to the resistant cultivar MR-1 indicated that a gene conversion had occurred in Fom-2, resulting in a significant rearrangement of this gene. The second candidate gene which shared high similarity to an essential gene in Arabidopsis, presented an almost identical sequence in MR-1 and BL, further supporting Fom-2 identity. The gene conversion in Fom-2 produced a truncated R gene, revealing new insights into R gene evolution. Fom-2 was predicted to encode an NBS-LRR type R protein of the non-TIR subfamily. In contrast to most members of this class a coiled-coil structure was predicted within the LRR region rather than in the N-terminal. The Fom-2 physical region contained retroelement-like sequences and truncated genes, suggesting that this locus is complex.
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.
An eight-cysteine-containing CFEM domain unique to a group of fungal membrane proteins.
CFEM, an eight cysteine-containing domain, has been identified by analyzing over 25 fungal sequences selected from database sequence searches. Features of CFEM suggest that it is a novel domain with characteristics distinct from known cysteine-rich domains. Some CFEM-containing proteins (e.g. Pth11 from Magnaporthe grisea) are proposed to have important roles in fungal pathogenesis.
AFLP analysis of a worldwide collection of Didymella bryoniae.
Didymella bryoniae (anamorph Phoma cucurbitacearum) is an ascomycete that causes gummy stem blight, a foliar disease that occurs on cucurbits in greenhouses and fields throughout the world. In a previous study using RAPD analysis, little genetic diversity was found among isolates of D. bryoniae from New York and South Carolina, USA. Here we report the use of amplified fragment length polymorphism (AFLP) analysis to assess the genetic variation within a worldwide collection of D. bryoniae, 102 field and greenhouse isolates from ten states in the USA (California, Delaware, Florida, Georgia, Indiana, Maryland, Michigan, Oklahoma, South Carolina, and Texas) and seven other countries (Australia, Canada, China, Greece, Israel, Sweden, and The Netherlands) were examined. Seven different AFLP primer-pair combinations generated 450 bands, of which 134 were polymorphic (30%). Using cluster analysis, two groups and a total of seven subgroups were delineated. Representative isolates varied in their virulence on muskmelon and watermelon seedlings, but the degree of virulence was not strongly associated with AFLP groupings. However, isolates from the northern USA grouped separately from isolates originating from the southern USA.
MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea.
We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.
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.
An integrated physical and genetic map of the rice genome.
Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome ( approximately 90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.