Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina.

The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.

Maintaining two mating types: structure of the mating type locus and its role in heterokaryosis in Podospora anserina.

Pseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat- isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat- mating types of P. anserina, which is mostly found in nature as a mat+/mat- heterokaryotic mycelium harboring sexually compatible nuclei. We identified a "mat" region ∼0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat- strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat- chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat-, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat-/mat- heterokaryons are as stable as mat+/mat- ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat- strains.

A network of HMG-box transcription factors regulates sexual cycle in the fungus Podospora anserina.

High-mobility group (HMG) B proteins are eukaryotic DNA-binding proteins characterized by the HMG-box functional motif. These transcription factors play a pivotal role in global genomic functions and in the control of genes involved in specific developmental or metabolic pathways. The filamentous ascomycete Podospora anserina contains 12 HMG-box genes. Of these, four have been previously characterized; three are mating-type genes that control fertilization and development of the fruit-body, whereas the last one encodes a factor involved in mitochondrial DNA stability. Systematic deletion analysis of the eight remaining uncharacterized HMG-box genes indicated that none were essential for viability, but that seven were involved in the sexual cycle. Two HMG-box genes display striking features. PaHMG5, an ortholog of SpSte11 from Schizosaccharomyces pombe, is a pivotal activator of mating-type genes in P. anserina, whereas PaHMG9 is a repressor of several phenomena specific to the stationary phase, most notably hyphal anastomoses. Transcriptional analyses of HMG-box genes in HMG-box deletion strains indicated that PaHMG5 is at the hub of a network of several HMG-box factors that regulate mating-type genes and mating-type target genes. Genetic analyses revealed that this network also controls fertility genes that are not regulated by mating-type transcription factors. This study points to the critical role of HMG-box members in sexual reproduction in fungi, as 11 out of 12 members were involved in the sexual cycle in P. anserina. PaHMG5 and SpSte11 are conserved transcriptional regulators of mating-type genes, although P. anserina and S. pombe diverged 550 million years ago. Two HMG-box genes, SOX9 and its upstream regulator SRY, also play an important role in sex determination in mammals. The P. anserina and S. pombe mating-type genes and their upstream regulatory factor form a module of HMG-box genes analogous to the SRY/SOX9 module, revealing a commonality of sex regulation in animals and fungi.

Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

The transcriptional response to the inactivation of the PaMpk1 and PaMpk2 MAP kinase pathways in Podospora anserina.

Transcription pattern during mycelium growth of Podospora anserina was assayed by microarray analysis in wild type and in mutants affected in the MAP kinase genes PaMpk1 and PaMpk2 and in the NADPH oxidase gene PaNox1. 15% of the genes have their expression modified by a factor two or more as growth proceeds in wild type. The genes whose expression is modified during growth in P. anserina are either not conserved or differently regulated in Neurospora crassa and Aspergillus niger, two fungi for which transcriptome data during growth are available. The P. anserina mutants display a similar alteration of their transcriptome profile, with nearly 1000 genes affected similarly in the three mutants, accounting for their similar growth phenotypes. Yet, each mutant has its specific set of modified transcripts, in line with particular phenotypes exhibited by each mutant. Again, there is limited conservation during evolution of the genes regulated at the transcription level by MAP kinases, as indicated by the comparison the P. anserina data, with those of Aspergillus fumigatus and N. crassa, two fungi for which gene expression data are available for mutants of the MAPK pathways. Among the genes regulated in wild type and affected in the mutants, those involved in carbohydrate and secondary metabolisms appear prominent. The vast majority of the genes differentially expressed are of unknown function. Availability of their transcription profile at various stages of development should help to decipher their role in fungal physiology and development.

The importomer peroxins are differentially required for peroxisome assembly and meiotic development in Podospora anserina: insights into a new peroxisome import pathway.

Peroxisome biogenesis relies on two known peroxisome matrix protein import pathways that are mediated by the receptors PEX5 and PEX7. These pathways converge at the importomer, a peroxisome-membrane complex that is required for protein translocation into peroxisomes and consists of docking and RING-finger subcomplexes. In the fungus Podospora anserina, the RING-finger peroxins are crucial for meiocyte formation, while PEX5, PEX7 or the docking peroxin PEX14 are not. Here we show that PEX14 and the PEX14-related protein PEX14/17 are differentially involved in peroxisome import during development. PEX14/17 activity does not compensate for loss of PEX14 function, and elimination of both proteins has no effect on meiocyte differentiation. In contrast, the docking peroxin PEX13, and the peroxins implicated in peroxisome membrane biogenesis PEX3 and PEX19, are required for meiocyte formation. Remarkably, the PTS2 coreceptor PEX20 is also essential for meiocyte differentiation and this function does not require PEX5 or PEX7. This finding suggests that PEX20 can mediate the import receptor activity of specific peroxisome matrix proteins. Our results suggest a new pathway for peroxisome import, which relies on PEX20 as import receptor and which seems critically required for specific developmental processes, like meiocyte differentiation in P. anserina.

Genome-wide gene expression profiling of fertilization competent mycelium in opposite mating types in the heterothallic fungus Podospora anserina.

BACKGROUND: Mating-type loci in yeasts and ascomycotan filamentous fungi (Pezizomycotina) encode master transcriptional factors that play a critical role in sexual development. Genome-wide analyses of mating-type-specification circuits and mating-type target genes are available in Saccharomyces cerevisiae and Schizosaccharomyces pombe; however, no such analyses have been performed in heterothallic (self-incompatible) Pezizomycotina. The heterothallic fungus Podospora anserina serves as a model for understanding the basic features of mating-type control. Its mat+ and mat- mating types are determined by dissimilar allelic sequences. The mat- sequence contains three genes, designated FMR1, SMR1 and SMR2, while the mat+ sequence contains one gene, FPR1. FMR1 and FPR1 are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptomic profiles of the mat+ and mat- strains revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1(-) and fpr1(-) mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species. CONCLUSIONS/SIGNIFICANCE: This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating.

The genome sequence of the model ascomycete fungus Podospora anserina.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: The 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.

Gene deletion and allelic replacement in the filamentous fungus Podospora anserina.

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the DeltaPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina.

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus '14', called segment '29'. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.

The function of the coding sequences for the putative pheromone precursors in Podospora anserina is restricted to fertilization.

We cloned the pheromone precursor genes of Podospora anserina in order to elucidate their role in the biology of this fungus. The mfp gene encodes a 24-amino-acid polypeptide finished by the CAAX motif, characteristic of fungal lipopeptide pheromone precursors similar to the a-factor precursor of Saccharomyces cerevisiae. The mfm gene encodes a 221-amino-acid polypeptide, which is related to the S. cerevisiae alpha-factor precursor and contains two 13-residue repeats assumed to correspond to the mature pheromone. We deleted the mfp and mfm coding sequence by gene replacement. The mutations specifically affect male fertility, without impairing female fertility and vegetative growth. The male defect is mating type specific: the mat+ Deltamfp and mat- Deltamfm mutants produce male cells inactive in fertilization whereas the mat- Deltamfp and mat+ Deltamfm mutants show normal male fertility. Genetic data indicate that both mfp and mfm are transcribed at a low level in mat+ and mat- vegetative hyphae. Northern-blot analysis shows that their transcription is induced by the mating types in microconidia (mfp by mat+ and mfm by mat-). We managed to cross Deltamfp Deltamfm strains of opposite mating type, by complementation and transient expression of the pheromone precursor gene to trigger fertilization. These crosses were fertile, demonstrating that once fertilization occurs, the pheromone precursor genes are unnecessary for the completion of the sexual cycle. Finally, we show that the constitutively transcribed gpd::mfm and gpd::mfp constructs are repressed at a posttranscriptional level by the noncognate mating type.

The fle1 gene encoding a C2H2 zinc finger protein co-ordinates male and female sexual differentiation in Podospora anserina.

The flexuosa (fle1-1) mutant, isolated in Podospora anserina, displays vegetative defects and two antagonistic sexual phenotypes: it produces several 1000-fold fewer microconidia (male gametes) than the wild-type strain and, conversely, more abundant protoperithecia (female organs). Cloning and sequencing of the fle1 gene and of cDNA identified an open reading frame encoding a 382-amino-acid polypeptide with two C2H2 zinc finger motifs. The predicted FLE1 protein shares 46% identity with the FlbC protein of Aspergillus nidulans and 68% identity with a putative protein identified by a search in the Neurospora crassa database. The nuclear localization of FLE1 was demonstrated by fusion with the green fluorescent protein. Sequencing of the fle1-1 mutant allele revealed a frameshift mutation upstream of the zinc finger domain. The fle1-1 mutant was a null mutant, as targeted disruption of fle1 sequence led to the same pleiotropic phenotype. When fle1 was overexpressed by introduction of a transgenic copy of the native fle1 gene or a fusion with a strong promoter, formation of protoperithecia was impaired, leading to partial or complete female sterility. We propose that fle1 acts as a repressor of female sexual differentiation in order to maintain the balance between male and female sexual pathways.