Sylvie Arnaise

The genome sequence of the model ascomycete fungus Podospora anserina

BackgroundThe dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.ResultsWe present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown.ConclusionThe features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.

The mat-- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs

In the filamentous fungus Podospora anserina, mating type is specified by a single locus with two alternate alleles, termed mat- and mat+. A previous study has shown that the mat+ sequence consists of 3.7 kb and contains a single gene relevant to the sexual cycle. This gene, called FPR1, encodes a protein with a HMG DNA-binding domain and is required for fertilization and for the development of the fertilized fruiting body. The mat-sequence, which is 4.7 kb in length, displays a more complex structure. We present here the characterization of two genes, called SMR1 and SMR2, which are present in the mat- allele along with the FMR1 gene. FMR1, whose role in the sexual cycle has been already partially described, encodes a protein with an α1-domain and was shown to control fertilization. We demonstrate that these three genes are required for the developmental events that occur in the female organ after fertilization. The additional role of FMR1 requires a region of unknown function that is distinct from the α1-domain. SMR1 encodes a protein with a putative acidic/hydrophobic α-helix, which has been proposed to be a feature common to transcriptional activators. The protein sequence deduced from SMR2 contains an HMG motif suggesting that it is a transcription factor.

Comparative genomics of emerging pathogens in the Candida glabrata clade

BackgroundCandida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts.ResultsOur results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata.ConclusionsRemarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces.Sequence Accession NumbersNakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

Abstract In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat− or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins.

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat−, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat− information. The transformants behave in crosses as do the reference mat+ or mat− strains, thus indicating that the transgenic mat+ and mat− are fully functional even when they have integrated at ectopic sites.

pah1: a homeobox gene involved in hyphal morphology and microconidiogenesis in the filamentous ascomycete Podospora anserina

Homeobox‐containing genes are widely described among eukaryotic species other than filamentous ascomycetes. We describe here the isolation and characterization of the first homeobox gene (pah1) identified in a filamentous ascomycete. It encodes a putative protein of 610 amino acids containing a typical homeodomain with 60 amino acids. Deletion of the pah1 gene enhances the number of male gametes (microconidia), whereas overexpression of pah1 results in a decrease in microconidia. These results led us to suppose that pah1 may be a repressor of genes involved in the microconidiation process. Moreover, pah1 is involved in hyphal branching and possibly in the development of female organs.

Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina.

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death.

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.