Abstract not available

Gillian Turgeon, Jinyuan Liu, Banu Metin

In filamentous ascomycetes, mating type loci exist as two alternate forms, MAT1-1 and MAT1-2, defining two mating types. While MAT1-2 contains a single gene encoding a protein with an HMG box domain in all ascomycetes examined so far, MAT1-1 shows differences. Zygomycetes encode an HMG type protein at both idiomorphs. In Sordariomycetes, the MAT1-1 idiomorph contains three genes: MAT1-1-1 encoding a protein with an alpha box domain, MAT1-1-2 encoding a protein with an HPG domain, and MAT1-1-3 encoding a protein with an HMG box domain. In Dothideomycetes, the MAT1-1 idiomorph harbors a single gene, encoding a protein with an alpha box domain. Careful comparison of the MAT1-1-1 and MAT1-2-1 proteins, however, reveals conserved motifs, one of which resembles an HMG box, in MAT1-1-1, as well as in MAT1-2-1. To determine if this second HMG motif is functionally important, point mutations were made in Cochliobolus heterostrophus MAT1-2-1 sequences, as controls, and in the corresponding positions of MAT1-1-1 sequences. Mutated genes and wild-type MAT gene controls were used to transform a MAT-deleted C. heterostrophus strain and the strains tested for mating competence. Effects of the mutations on mating ability address the question of how various ascomycete MAT loci have evolved to carry out mating activities.

Matthew Fisher¹, Trent Garner², David Aanensen¹

1Imperial College, London, United Kingdom, ²Institute of Zoology, London, United Kingdom

Detailed multilocus molecular typing is now regularly used to describe the population genetic structure of mycoses, leading to insights into their principle modes of transmission, their environmental reservoirs, and their longer-term evolution. In parallel, developments in bioinformatics, spatial analyses and emerging web technologies allow globally accessible multilocus typing databases to be merged with powerful spatial mapping tools in realtime. Here, we describe the development of a community-focused mapping tool, www.spatialepidemiology.net/bd, that allows the spatial population genetic structure of B. dendrobatidis to be analysed within the species global occurrence. We show, using the European Union B. dendrobatidis surveillance database, that the organism is widespread across Europe however is spatially heterogeneous at smaller scales. We demonstrate, using multilocus genotype data, that the amphibian trade and amphibian re-introduction programs have lead to the recent introduction of B. dendrobatidis in several regions. Bayesian statistical analysis and mathematical models paramaterised using the European Union database show that there is significant heterogeneity in the risk of infected species developing chytridiomycosis, and we show that environmental variables are associated with the risk of developing disease. We argue that this spatial bioinformatic approach to analysing the emergence of fungal disease has wide applicability, and illustrate this using the Cryptococcus neoformans mlst database http://cneoformans.mlst.net/.

Stefanie Pöggeler¹, Birgit Hoff², Ulrich Kück²

1Georg-August Universtity Göttingen, Institute of Microbiology and Genetics, Göttingen, Germany, ²Ruhr-University Bochum, LS General and Molecular Botany, Bochum, Germany

The ubiquitous genus Penicillium consists of numerous important, apparently asexual species. However, analysis of the complete genome sequences of the asexual human pathogens Aspergillus fumigatus and Penicillium marneffei, revealed the presence of genes associated with sexual reproduction, including mating-type genes and genes for pheromone production and detection.

The APN2 gene encoding a putative DNA lyase and the SLA2 gene encoding a cytoskeleton assembly control factor have been reported neighbouring MAT loci in many ascomycetes. Therefore, to determine whether mating-type genes are also present in the industrially relevant fungus Penicillium chrysogenum, we designed primers corresponding to conserved regions of the A. fumigatus APN2 and SLA2 genes, respectively. Using genomic DNA from twelve different P. chrysogenum isolates two different types of amplicons were obtained. Six isolates generated a larger amplicon, while the other six, generated a smaller one. Sequencing of the larger amplicon revealed the presence of a putative MAT-1 gene and sequencing of the smaller amplicon revealed homology to a putative MAT2 gene. Similar to the structural organization of mating-type loci in sexual reproducing filamentous ascomycetes, highly conserved flanking regions were found upstream and downstream of the P. chrysogenum MAT idiomorphs. RT-PCR analyses showed that mating-type genes are expressed and suggest that P. chrysogenum has the potential to reproduce sexually. This finding prompted us to search for homologs of pheromone and pheromone receptor genes that function in mating and pheromone signalling in sexual reproducing filamentous ascomycetes. Indeed, a screen of a P. chrysogenum cDNA library led to the isolation of a putative P. chrysogenum gene encoding a peptide pheromone precursor and two pheromone receptor genes. Using an RT-PCR approach, we demonstrated that both pheromone and receptor genes are expressed in strains of both mating types. The results of our transcriptional expression data suggest the existence of a sexual cycle in P. chrysogenum. This finding opens up the possibility to induce mating and sexual reproduction in P. chrysogenum.

Magnus Karlsson, Jan Stenlid

Gene duplication and loss play an important role in the evolution of novel functions and for shaping an organism’s gene content. Recently, it was suggested that stress-related genes frequently are exposed to duplications and losses, while growth-related genes show selection against change in copy number. The fungal chitinase gene family constitutes an interesting case study of gene duplication and loss, as their biological roles include growth and development as well as more stress-responsive functions. We used genome sequence data to analyze the size of the chitinase gene family in different fungal taxa, which range from 1 in Batrachochytrium dendrobatidis and Schizosaccharomyces pombe to 20 in Hypocrea jecorina and Emericella nidulans, and to infer their phylogenetic relationships. Novel chitinase subgroups are identified and their phylogenetic relationships with previously known chitinases are discussed. Subgroups can have a specific domain-structure that suggests functional adaptations. We also employ a stochastic birth and death model to show that the fungal chitinase gene family indeed evolves non-randomly, and we identify six fungal lineages where larger-than-expected expansions (Pezizomycotina, H. jecorina, Gibberella zeae, Uncinocarpus reesii, E. nidulans and Rhizopus oryzae), and two contractions (Coccidioides immitis and S. pombe) potentially indicate the action of adaptive natural selection. Expansions and contractions of specific chitinase subgroups putatively involved in antagonistic fungal-fungal interactions and selection against change in copy number in other chitinase subgroups involved in growth and development are discussed in relation to the ecological role and lifestyle of different fungal taxa.

Anne D. van Diepeningen, Alfons J.M. Debets, S. Marijke Slakhorst, Rolf F. Hoekstra

Calorie restriction (CR) refers to a dietary regimen low in calories without starvation. CR extends the life span of many organisms, varying from yeasts to vertebrates and possibly even nonhuman primates. The presumed adaptive value of calorie restriction is that it allows organisms to postpone reproduction until food is available. But, although CR has been known for more than 70 years to extend life span, the molecular basis is so far unknown.

Podospora anserina is one of the few filamentous fungi known to age in nature. It can be grown under controlled conditions on synthetic medium, has a life span of approximately 2-3 weeks and thanks to years of research quite a lot is known about ageing in this species, making it an ideal model organism.

We show that also the filamentous fungus Podospora anserina grown on a controlled synthetic medium has a longer life span under CR, either with glucose, fructose or acetate as substrate. Several mechanisms for the CR response have been proposed: Our results rule out two of these: neither relief of glucose toxicity nor catabolite repression explains the observed CR effects. Furthermore, CR increased also the reproductive life span and the mitochondrial stability. Rearrangements in the so-called alpha senDNA region of the mitochondrial genome, typically observed during ageing under nutrient rich conditions, were reduced or absent under CR. CR extended the period of self-fertility. In addition, after a period on the lowest tested calorie level, fungal lines lost part of their mating type information, turning them from inbreeding pseudohomothallic to outbreeding heterothallic strains.

Mathieu Paoletti¹, Eric Bastiaans², Damien Chevanne¹, Pal Karoly², Sven Saupe¹, Fons Debets², Corinne Clavé¹

1Laboratoire de Génétique Moléculaire des Champignons, IBGC, UMR 5095 Université Bordeaux2 et CNRS, Bordeaux, France, ²Laboratory of Genetics, Wageningen University, Wageningen, Netherlands

Non self recognition leading to vegetative incompatibility (VI) is a ubiquitous phenomenon in filamentous fungi presumed to restrict cellular parasitism as well as transmission of deleterious cytoplasmic elements. Vegetative non self recognition is determined by specific het genes that decide the fate of heterokaryons produced after fusion of cells of unlike genotypes. Expression of incompatible alleles at any het locus triggers VI and result in cell death of the fusion cell. het genes are unusually polymorphic and unravelling mechanisms for genesis/maintenance of polymorphism is key to understanding VI.

In Podospora anserina, co-expression of incompatible het-C/het-D or het-C/het-E alleles results in VI. het-C encodes for a Glycolipid Transfer Protein (GLTP), and het-D and het-E are paralogues encoding for three domain proteins with an N-terminal HET domain mediating cell death, a central NACHT domain binding to GTP, and a variable C-terminal WD domain whose sequence defines allelic specificity. The WD domain is made of tandem repeats of WD-40 units that assemble in a beta propeller fold involved in protein-protein interactions. Genome sequence analysis revealed that het-D and het-E belong to a large gene family called NWD. We investigate evolution of the components of these non self recognition systems.

We identified three evolutionary forces acting on the WD-40 sequences whose combination promotes fast evolution of the WD recognition domain : Repeat Induced Point Mutation introduces mutations in the WD-40 repeat unit sequences, concerted evolution allows for WD-40 unit exchanges between NWD loci, and positive selection retains favourable variants located in the protein-protein interaction surface of the beta propeller fold (WD domain). We also demonstrate that newly discovered members of the NWD gene family are incompatibility genes. Finally, we identified numerous as yet unknown het-C alleles that display new incompatibility interaction patterns with het-D and het-E, and demonstrate that het-C gene is under positive diversifying selection while fungal GLTP encoding genes as a whole are under purifying selection. Overall this dynamic system appears to promote HNWD and het-C co-evolution to promote the appearance of new VI systems.

Michael Fedler, Michael Mahlert, Fernanda Nieto, Kai-Steffen Luh, Kathrin Stelter, Christoph W. Basse

We have investigated a role of the Ustilago maydis a2 mating type locus genes lga2 and rga2 in uniparental mitochondrial inheritance during sexual development in the host plant maize. The lga2 and rga2 genes encode small mitochondrial proteins that are dispensable for pathogenic development. Based on the identification of different mitochondrial DNA (mtDNA) genotypes and the generation of U. maydis mutant strains individually deleted in either lga2 or rga2, we could demonstrate that these genes decisively influence the outcome of uniparental mitochondrial inheritance. Their deletion either reverted the outcome of mitochondrial transmission or led to biparental inheritance and exchange between parental mtDNAs. In addition, we could provide evidence that homing endonuclease genes are involved in the generation of new mitotypes during sexual development. Our results show for the first time a direct role of mitochondrial proteins in regulating uniparental mitochondrial inheritance and suggest that Lga2/Rga2 function as two-component system to mediate this process. We additionally provide evidence that Lga2 interferes with the mitochondrial fusion/fission machinery to prevent mitochondrial fusion and thus limit opportunity for mtDNA exchange.