Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with the formation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defect in ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia and carries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequence analyses revealed three putative transmembrane domains and a C-terminal domain of unknown function. Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolar network of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from the large spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.The formation of fruiting bodies in filamentous ascomycetes is a process of multicellular differentiation controlled by many developmentally regulated genes. The self-fertile ascomycete Sordaria macrospora represents an excellent model for studying cell differentiation during fungal fruiting body development (reviewed in references 9 and 24).During the life cycle of S. macrospora, a mature haploid ascospore germinates and produces a mycelium composed of multinucleate hyphal compartments. After 2 to 3 days, coiled female reproductive hyphae, called ascogonia, are formed. Each ascogonium develops further into a more-or-less spherical protoperithecium composed of pseudoparenchymatous tissue surrounding the original ascogonium (24, 47). Ascogenous hyphae emerge from the ascogonium inside the protoperithecium. Within the ascogenous hyphae, the nuclei pair up to form the dikaryotic state, even though S. macrospora is selffertile and does not require fertilization with an opposite mating type (11,49). The transition from the spherical protoperithecial to the flask-shaped perithecial stage, is believed to be stimulated by the formation of the dikaryon, although this has not been experimentally verified (24). The dikaryotic state in individual hyphal compartments of the growing ascogenous hyphae is maintained by precisely orchestrated crozier formation from the tip of an ascogenous hypha, fusion of the crozier tip with the penultimate cell of the ascogenous hypha, conjugate mitotic divisions, and highly regulated septation (50, 69). Karyogamy of two nuclei in the penultimate cell results in the formation of a diploid ascus mother cell. The diploid state is very short-lived because the diploid nuclei which are formed immediately undergo meiosis, followed by an extra mitotic division resulting in an ascus containing eight haploid ascospores (11,49). An identical process has been observed in the heterothallic fungus Neurospora crassa, with the exception that cell fusion between opposit...

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.Sexual development in filamentous ascomycetes is usually accompanied by the formation of complex fruiting bodies. During this process, a number of specialized cell types have to be generated from a more or less undifferentiated vegetative mycelium (5). This morphogenetic program is a convenient system that allows us to address the following fundamental question: how is multicellular development coordinated in eukaryotes? So far, several environmental and endogenous signals have been described to regulate fruiting body-dependent gene expression in a temporal and spatial manner (for a review, see reference 55). However, even though a decent number of genes involved in this process have been identified, the molecular determinants of fruiting body development have yet to emerge.In this study, we have investigated the homothallic ascomycete Sordaria macrospora, which was recently developed as a model system to study fruiting body morphogenesis (27,36,37,43,52). During early sexual propagation, S. macrospora forms female gametangia, so-called ascogonia, on vegetative hyphae. In subsequent differentiation processes, ascogonia are enveloped by sterile hyphae and develop into spherical premature structures (protoperithecia), which mature into flask-like fruiting bodies (perithecia). In its final developmental stage, each perithecium contains a set of 60 to 80 asci with eight linearly ordered ascospores.Using a forward genetic approach, we aimed to identify numerous developmental genes essential for fruiting body development in S. macrospora. Using conventional mutagenesis, several developmental mutants defective at different stages of perithecium formation were generated (37). In recent years,...

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The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.