Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization

Comparative Genomics and Transcriptomics To Analyze Fruiting Body Development in Filamentous Ascomycetes.

Many filamentous ascomycetes develop three-dimensional fruiting bodies for production and dispersal of sexual spores. Fruiting bodies are among the most complex structures differentiated by ascomycetes; however, the molecular mechanisms underlying this process are insufficiently understood. Previous comparative transcriptomics analyses of fruiting body development in different ascomycetes suggested that there might be a core set of genes that are transcriptionally regulated in a similar manner across species. Conserved patterns of gene expression can be indicative of functional relevance, and therefore such a set of genes might constitute promising candidates for functional analyses. In this study, we have sequenced the genome of the Pezizomycete Ascodesmis nigricans, and performed comparative transcriptomics of developing fruiting bodies of this fungus, the Pezizomycete Pyronema confluens, and the Sordariomycete Sordaria macrospora With only 27 Mb, the A. nigricans genome is the smallest Pezizomycete genome sequenced to date. Comparative transcriptomics indicated that gene expression patterns in developing fruiting bodies of the three species are more similar to each other than to nonsexual hyphae of the same species. An analysis of 83 genes that are upregulated only during fruiting body development in all three species revealed 23 genes encoding proteins with predicted roles in vesicle transport, the endomembrane system, or transport across membranes, and 13 genes encoding proteins with predicted roles in chromatin organization or the regulation of gene expression. Among four genes chosen for functional analysis by deletion in S. macrospora, three were shown to be involved in fruiting body formation, including two predicted chromatin modifier genes

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

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 Mating-Type Chromosome in the Filamentous Ascomycete Neurospora tetrasperma Represents a Model for Early Evolution of Sex Chromosomes

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first “evolutionary stratum”, genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers

Abstracts from the Neurospora 2004 conference

Abstracts from the Neurospora 2004 conferenc

The genetics of circadian rhythms in Neurospora crassa : do multiple oscillators exist?

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Includes bibliographical references (leaves 19-21).Circadian biological clocks are found in virtually all organisms and function to generate daily rhythms. The circadian clock (built by one or more oscillators) controls rhythmicity in a wide range of processes, ranging from sleep/wake cycles in humans, photosynthesis in some plants, to reproduction in fungus and other eukaryotes. One organism, Neurospora crassa, displays an easily observed and assayable circadian rhythm in asexual spore production, conidiation, and is an excellent model for understanding the molecular and biochemical basis of circadian rhythms. Studies in Neurospora have identified the frequency (frq) gene as a central component of the fungal clock. Under most growth conditions, rhythmic development is absent in strains lacking a functional FRQ protein. However, under some conditions, rhythmic conidiation can be observed in a FRQ-null strain. This residual rhythmicicty was noticed early on, but went essentially ignored until recent experiments demonstrated rhythms in cultured FRQ-null strains grown in 12 hour temperature cycles in constant darkness. These data suggested that the FRQ-based oscillator is not the only oscillator in the cell and led to our hypothesis that the clock system is composed of more than one oscillator that can function to generate rhythms. To test this hypothesis, I have created random mutations in a FRQ-null strain and assayed for loss of rhythmicity in the temperature cycles. Fourteen mutant strains were identified that met these criteria. It is expected that some of these mutations will identify genes that function in the temperature-dependent oscillator

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum

Identification Of Novel Nuclear Proteins Required For Meiotic Silencing By Unpaired Dna In Neurospora Crassa

A fundamental step that occurs during sexual reproduction is meiosis, which is a specialized type of cell division. During meiosis, pairs of chromosomes exchange genetic information via recombination. At this point, the genome is particularly susceptible to viruses and other foreign genetic invasions. Therefore, it is important to protect the genome to prevent the transmission of foreign genetic materials to the offspring. There are several mechanisms work together to protect host genome from foreign genetic materials. These are known as “genome defense mechanisms”. The fungus Neurospora crassa is one of the best organisms for genome defense studies due to the presence of at least three genome defense mechanisms; including Repeat Induced Point mutation (RIP), Quelling, and Meiotic Silencing by Unpaired DNA (MSUD).The main focus of my dissertation is the MSUD pathway. MSUD is a process that detects and silences unpaired DNA between homologous chromosomes. During MSUD, Homologous chromosomes are scanned for unpaired regions by unknown protein complexes. These protein complexes may also contribute to homology search required by some DNA repair pathways. Therefore, identification of these proteins could thus have a significant impact for cancer research. Hence, one part of my dissertation is to identify and characterize novel proteins that detect unpaired DNA during meiotic silencing. In my findings, I have found a putative SNF2-family protein (SAD-6) required for efficient MSUD in Neurospora crassa and it is closely related to a protein called Rad54, which involved in the repair of DNA double-strand breaks by homologous recombination. Moreover, I was able to identify and characterize Neurospora crassa sad-7, a gene encoding a protein with RNA recognition motif (RRM). My experiments have confirmed that SAD-7 in N. crassa, is required for fully-efficient MSUD in the presence of unpaired DNA. Additionally, I have focused on Meiotic drive elements. These elements are found in eukaryotic genomes. In general, genetic loci are transmitted to the offspring during sexual reproduction by following the Mendelian inheritance patterns. However, there are some selfish loci that are capable of bias their own transmission rates through meiosis or during gametogenesis in the presence of a competing locus. These are known as meiotic drive elements. Neurospora crassa has a meiotic drive element known as Spore killer-2 (Sk-2) and it achieves the biased transmission by spore killing. When Sk-2 is crossed into a Spore killer sensitive opposite mating type (SkS), hypothetically there should be a mixed offspring population of killer resistant and killer sensitive ascospores. Surprisingly, when analyzing the ascospores, nearly all the survived ascospores express the Sk-2 genotype and all the ascospores with the Spore killer sensitive genotype are non-viable. However, there are a little known about the exact location of Sk-2 meiotic drive element and it’s mechanism of transmission. In my experiments, I was able identify a genetic element located in Neurospora chromosome III that is required and sufficient for spore killing. Overall, my results provide new insights to the search and unpaired DNA detection during meiosis and also the identification of the genetic element required for spore killing sheds lights towards the understanding of the spore killer mechanism in Neurospora crassa

Neurospora Bibliography

This bibliography represents my attempt to collect all works dealing substantially with Neurospora