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The development of mycological gerontology requires effective methods for assessing the biological age of fungal cells. This assessment is based on the analysis of a complex of aging and oxidative stress markers. One of the most powerful such markers is the protein carbonylation. In this study, the already known method of dry immune dot blotting is adapted for mycological studies of the content of protein carbonyl groups. After testing the method on a number of filamentous fungi species, some features of the accumulation of carbonylated proteins in mycelium were established. Among these features: (i) a weak effect of exogenous oxidative stress on the accumulation of carbonyls in a number of fungi, (ii) reversibility of the carbonyl accumulation, (iii) possibility of arbitrary regulation of carbonyl content by fungus itself and (iv) the influence of hormesis. In addition, two polar strategies for the accumulation of carbonyl modification were revealed, named Id-strategy (Indifferent) and Cn-strategy (Concern). Thus, even the analysis of one marker allows making some preliminary general assumptions and conclusions. For example, the idea that fungi can freely regulate their biological age is confirmed. This feature makes fungi very flexible in terms of responding to environmental influences and promising objects for gerontology.

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first 4 years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, in the G-protein alpha subunit FadA, new alleles fixed in seven out of eight experimental populations, and these fixations affected just four amino acid sites, which is an unprecedented level of parallelism in experimental evolution. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed fixations were adaptive and driven by positive selection.

Filamentous fungi are known as prolific untapped reservoirs of diverse secondary metabolites, where genes required for their synthesis are organized in clusters. The bioactive properties of these compounds are closely related to their functions in fungal biology, which are not well understood. In this study, we focused on the Podospora anserina gene cluster responsible for the biosynthesis of sterigmatocystin (ST). Deletion of the PaStcA gene encoding the polyketide synthase and overexpression (OE) of the PaAflR gene encoding the ST-specific transcription factor in P. anserina were performed. We showed that growth of PaStcAΔ was inhibited in the presence of methylglyoxal, while OE-PaAflR showed a little inhibition, indicating that ST production may enhance oxidative stress tolerance in P. anserina. We also showed that the OE-PaAflR strain displayed an overpigmented thallus mediated by the melanin pathway. Overexpression of PaAflR also led to sterility. Interspecific confrontation assays showed that ST-overexpressed strains produced a high level of peroxides and possessed a higher competitiveness against other fungi. Comparative metabolite profiling demonstrated that PaStcAΔ strain was unable to produce ST, while OE-PaAflR displayed a ST overproduction. This study contributes to a better understanding of ST in P. anserina, especially with regard to its involvement in fungal physiology.

Melanins are pigments used by fungi to withstand various stresses and to strengthen vegetative and reproductive structures. In Sordariales fungi, their biosynthesis starts with a condensation step catalyzed by an evolutionary-conserved polyketide synthase. Here we show that complete inactivation of this enzyme in the model ascomycete Podospora anserina through targeted deletion of the PaPks1 gene results in reduced female fertility, in contrast to a previously analyzed nonsense mutation in the same gene that retains full fertility. We also show the utility of PaPks1 mutants for detecting rare genetic events in P. anserina, such as parasexuality and possible fertilization and/or apomixis of nuclei devoid of mating-type gene.

Background Selective gene silencing is key to development. It is generally accepted that H3K27me3-enriched heterochromatin maintains transcriptional repression established during early development and regulates cell fate. Conversely, H3K9me3-enriched heterochromatin prevents differentiation but constitutes protection against transposable elements. We exploited the fungus Podospora anserina, a valuable alternative to higher eukaryote models, to question the biological relevance and functional interplay of these two distinct heterochromatin conformations. Results We established genome-wide patterns of H3K27me3 and H3K9me3 modifications, and found these marks mutually exclusive within gene-rich regions but not within repeats. We generated the corresponding histone methyltransferase null mutants and showed an interdependence of H3K9me3 and H3K27me3 marks. Indeed, removal of the PaKmt6 EZH2-like enzyme resulted not only in loss of H3K27me3 but also in significant H3K9me3 reduction. Similarly, removal of PaKmt1 SU(VAR)3–9-like enzyme caused loss of H3K9me3 and substantial decrease of H3K27me3. Removal of the H3K9me binding protein PaHP1 provided further support to the notion that each type of heterochromatin requires the presence of the other. We also established that P. anserina developmental programs require H3K27me3-mediated silencing, since loss of the PaKmt6 EZH2-like enzyme caused severe defects in most aspects of the life cycle including growth, differentiation processes and sexual reproduction, whereas loss of the PaKmt1 SU(VAR)3–9-like enzyme resulted only in marginal defects, similar to loss of PaHP1. Conclusions Our findings support a conserved function of the PRC2 complex in fungal development. However, we uncovered an intriguing evolutionary fluidity in the repressive histone deposition machinery, which challenges canonical definitions of constitutive and facultative heterochromatin.

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Most TEs studied to date are relatively small (50 – 12000 bp), but can contribute to very large proportions of genomes. Here we report the discovery of a giant tyrosine-recombinase-mobilized DNA transposon,Enterprise, from the model fungusPodospora anserina. Previously, we described a large genomic feature called theSpokblock which is notable due to the presence of meiotic drive genes of theSpokgene family. TheSpokblock ranges from 110 kb to 247 kb and can be present in at least four different genomic locations withinP. anserina, despite what is an otherwise highly conserved genome structure. We have determined that the reason for its varying positions is that theSpokblock is not only capable of meiotic drive, but is also capable of transposition. More precisely, theSpokblock represents a unique case where theEnterprisehas captured theSpoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate thatEnterprise(without theSpoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that theSpokblock is deleterious, with detrimental effects on spore production in strains which carry it. In contrast to the selfish role of theEnterpriseinP. anserina, we hypothesize that the mobility of theEnterprisemay also play an adaptive role in fungi whenEnterpriseundergoes horizontal transfer while carrying metabolic genes. This union of meiotic drivers and a transposon has created a selfish element of impressive size inPodospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

This chapter, discusses the unravel genetic mechanisms involved in the control of the life span of this simple eukaryotic microorganism. The extrachromosomal genetic trait controlling the onset of senescence has been identified in the mitochondria of senescent Podospora cultures. The experimental steps of such an analysis are: After mapping the gene of interest on a particular linkage group, cloning of the corresponding wild-type copy is possible by complementation of the long-lived mutant to wild-type characteristics using a genomic cosmid library of the wild-type strain. The frequency of postreduction depends on the distance of the corresponding genetic marker from the centromere, thus providing a basis for the construction of genetic maps. In the case of the mating-type locus, the two alleles undergo postreduction in about 98% of all cases since the mating-type locus is located rather on the end of linkage group 1.

Caseinolytic protease (CLPP) is an energy-dependent serine-type protease that plays a role in protein quality control. The CLPP gene is highly conserved across kingdoms and the protein is present in both bacteria and eukaryote organelles like mitochondria across a wide phylogenetic range. This pedigree has all the hallmarks of CLPP being an essential gene. However, in plants, disruption of mitochondrial CLPP has no impact on its growth, reminiscent of its nonessential role in some model fungi. Deletion of mitochondrial CLPP improves health and increased life span in the filamentous fungus, Podospora anserina, while loss of human mitochondrial CLPP leads to infertility and hearing loss. Recently it was revealed that both plant and human CLPP share a similar role in maintenance of the N-module of respiratory complex I. In addition, plant mitochondrial CLPP also coordinates the homeostasis of other mitochondrial protein complexes encoded by genes across mitochondrial and nuclear genomes. Understanding the contextual role of mitochondrial CLPP across kingdoms may help to understand these diverse sets of clpp phenotypes and the widespread conservation of CLPP genes.

SummaryS15/uS19 is one of the fifteen universally conserved ribosomal proteins of the small ribosomal subunit. While prokaryotic uS19 is located away from the mRNA decoding site, cross-linking studies identified eukaryotic uS19 C-terminal tail as contacting the A site on the 80S ribosome. Here, we study the effects of uS19 mutations isolated as translation accuracy mutations in the filamentous fungusPodospora anserina. All mutations alter residues of uS19 C-terminal tail, and cluster to the eukaryote-specific decapeptide 138-PGIGATHSSR-147. All mutations modify fungal development and cytosolic translation, albeit differently. Two mutations (P138S and S145F) increase fungus longevity and display mild effects on translation, while others (G139D and G139C) decrease longevity, have stronger effects on translation and confer hypersensitivity to paromomycin. By mimickingP. anserinamutations in the yeastSaccharomyces cerevisiae RPS15gene, we further show that P138S and S145F induce hyperaccurate recognition of the three stop codons, whereas G139D and G139C impair UAG and UAA codon recognition. Noteworthy, inP. anserina, uS19 genetically interacts with the eRF1 and eRF3 release factors. All together, our data indicate that uS19 C-terminal tail contributesin vivoto eukaryotic translation termination, and identify key amino acids of uS19 that potentially modulate eRF1-eRF3 interaction in the pre-termination complex.Graphical abstractAbbreviated SummaryS15/uS19 is a conserved small ribosomal protein that in eukaryotes harbors a flexible C-terminal extension proposed to interact with the A site mRNA codon during translation. Here, we describe how C-terminal variants variously affectPodospora anserinadevelopment and longevity and impact fungal ribosome and polysome formation. We reveal that stop codon recognition is significantly altered by the presence of those C-terminal variants, which either expand or on the contrary restrict termination ambiguity.

Plants and animals rely on cytoplasmic immune receptors of the NLR family to cope with a variety of biotic challenges. Proteins homologous to plant and animal NLRs were identified in fungi as part of the study of a non-self recognition and programmed cell death process known as heterokaryon incompatibility. We review the role of characterized NLR-type proteins in incompatibility in the three fungal species Podospora anserina, Cryphonectria parasitica, and Neurospora crassa and describe the phylogenetic distribution, domain architecture, and variability of the NLR gene repertoires in fungal genomes. We describe a specific type of NLR-mediated signal transduction process based on amyloid templating. We review the direct and indirect evidence suggesting that in general terms NLR-like proteins might also function as immune receptors in the fungal branch as they do in plant and animal lineages and discuss these implications in terms of the evolutionary trajectory of NLR genes.

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.

Background Autophagy is a conserved molecular pathway involved in the degradation and recycling of cellular components. It is active either as response to starvation or molecular damage. Evidence is emerging that autophagy plays a key role in the degradation of damaged cellular components and thereby affects aging and lifespan control. In earlier studies, it was found that autophagy in the aging model Podospora anserina acts as a longevity assurance mechanism. However, only little is known about the individual components controlling autophagy in this aging model. Here, we report a biochemical and bioinformatics study to detect the protein-protein interaction (PPI) network of P. anserina combining experimental and theoretical methods. Results We constructed the PPI network of autophagy in P. anserina based on the corresponding networks of yeast and human. We integrated PaATG8 interaction partners identified in an own yeast two-hybrid analysis using ATG8 of P. anserina as bait. Additionally, we included age-dependent transcriptome data. The resulting network consists of 89 proteins involved in 186 interactions. We applied bioinformatics approaches to analyze the network topology and to prove that the network is not random, but exhibits biologically meaningful properties. We identified hub proteins which play an essential role in the network as well as seven putative sub-pathways, and interactions which are likely to be evolutionary conserved amongst species. We confirmed that autophagy-associated genes are significantly often up-regulated and co-expressed during aging of P. anserina. Conclusions With the present study, we provide a comprehensive biological network of the autophagy pathway in P. anserina comprising PPI and gene expression data. It is based on computational prediction as well as experimental data. We identified sub-pathways, important hub proteins, and evolutionary conserved interactions. The network clearly illustrates the relation of autophagy to aging processes and enables further specific studies to understand autophagy and aging in P. anserina as well as in other systems. Electronic supplementary material The online version of this article (doi:10.1186/s12859-017-1603-2) contains supplementary material, which is available to authorized users.

Meiotic drive is the preferential transmission of a particular allele at a given locus during sexual reproduction. The phenomenon is observed as spore killing in a variety of fungal lineages, includingPodospora. In natural populations ofPodospora anserina, seven spore killers (Psks) have been identified through classical genetic analyses. Here we show that theSpokgene family underlie thePskspore killers. The combination of the variousSpokgenes at different chromosomal locations defines the spore killers and creates a killing hierarchy within the same population. We identify two novelSpokhomologs that are located within a complex region (theSpokblock) that reside in different chromosomal locations in given natural strains. We confirm that the individual SPOK proteins perform both the killing and resistance functions and show that these activities are dependent on distinct domains, a nuclease and a kinase domain respectively. Genomic data and phylogenetic analysis across ascomycetes suggest that theSpokgenes disperse via cross-species transfer, and evolve by duplication and diversification within several lineages.

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detectedin vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any.To do so, we used the model ascomycete fungusP. anserina. We identified thePaRidgene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRiddefective mutants. Crosses involvingP. anserinaΔPaRidmutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRidbackground, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRidmutants with ectopic alleles ofPaRid, including GFP-tagged, point-mutated, inter-specific and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.Author SummarySexual reproduction is considered to be essential for long-term persistence of eukaryotic species. Sexual reproduction is controlled by strict mechanisms governing which haploids can fuse (mating) and which developmental paths the resulting zygote will follow. In mammals, differential genomic DNA methylation patterns of parental gametes, known as ‘DNA methylation imprints’ are essential to zygotic development, while in plants, global genomic demethylation often results in female-sterility. Although animal and fungi are evolutionary related, little is known about epigenetic regulation of gene expression and development in multicellular fungi. Here, we report on a gene of the model fungusPodospora anserina, encoding a protein called PaRid that looks like a DNA methyltrasferase. We showed that expression of the catalytically functional version of the PaRid protein is required in the maternal parental strain to form zygotes. By establishing the transcriptional profile ofPaRidmutant strain, we identified a set of PaRid direct and/or indirect target genes. Half of them are also targets of a mating-type transcription factor known to be a major regulator of sexual development. So far, there was no other example of identified RID targets shared with a well-known developmental pathway that is common to an important group of fungi, the Pezizomycotina

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina., Author summary Sexual reproduction is considered to be essential for long-term persistence of eukaryotic species. Sexual reproduction is controlled by strict mechanisms governing which haploids can fuse (mating) and which developmental paths the resulting zygote will follow. In mammals, differential genomic DNA methylation patterns of parental gametes, known as ‘DNA methylation imprints’ are essential to zygotic development, while in plants, global genomic demethylation often results in female-sterility. Although animal and fungi are evolutionary related, little is known about epigenetic regulation of gene expression and development in multicellular fungi. Here, we report on a gene of the model fungus Podospora anserina, encoding a protein called PaRid that share several features with known DNA methyltransferases. We showed that expression of the catalytically functional version of the PaRid protein is required in the maternal parental strain to form zygotes. By establishing the transcriptional profile of PaRid mutant strain, we identified a set of PaRid target genes. Half of them are also targets of a mating-type transcription factor known to be a major regulator of sexual development. So far, there was no other example of identified RID targets shared with a well-known developmental pathway that is common to an important group of fungi, the Pezizomycotina.

International audience; By screening suppressors of a respiratory mutant lacking a functional cytochrome pathway in the filamentous fungus Podospora anserina, we isolated a mutation located in the phosphatase domain of the bi-functional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2). We show that the inactivation of the phosphatase but not of the kinase domain is responsible for the suppressor effect that results from the activation of the RSEs transcription factors that control expression of AOX, an alternative oxidase able to bypass the mitochondria cytochrome pathway of respiration. Remarkably, activation of the RSEs also stimulates the expression of the gluconeogenic enzymes, fructose-1,6 bi-phosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PCK-1). We thus reveal in P. anserina an apparently paradoxical situation where the inactivation of the phosphatase domain of PFK-2/FBPase-2, supposed to stimulate glycolysis, is correlated with the transcriptional induction of the gluconeogenic enzymes. Phylogenic analysis revealed the presence of multiple presumed PFK-2/FBPase-2 isoforms in all the species of tested Ascomycetes.

International audience; Fungi are prone to phenotypic instability, that is, the vegetative phase of these organisms, be they yeasts or molds, undergoes frequent switching between two or more behaviors, often with different morphologies, but also sometime having different physiologies without any obvious morphological outcome. In the context of industrial utilization of fungi, this can have a negative impact on the maintenance of strains and/or on their productivity. Instabilities have been shown to result from various mechanisms, either genetic or epigenetic. This chapter will review different types of instabilities and discuss some lesser-known ones, mostly in filamentous fungi, while it will direct readers to additional literature in the case of well-known phenomena such as the amyloid prions or fungal senescence. It will present in depth the "white/opaque" switch of Candida albicans and the "crippled growth" degeneration of the model fungus Podospora anserina. These are two of the most thoroughly studied epigenetic phenotypic switches. I will also discuss the "sectors" presented by many filamentous ascomycetes, for which a prion-based model exists but is not demonstrated. Finally, I will also describe intriguing examples of phenotypic instability for which an explanation has yet to be provided.

International audience; In filamentous fungi, entrance into stationary phase is complex as it is accompanied by several differentiation and developmental processes, including the synthesis of pigments, aerial hyphae, anastomoses and sporophores. The regulatory networks that control these processes are still incompletely known. The analysis of the "Impaired in the development of Crippled Growth (IDC)" mutants of the model filamentous ascomycete Podospora anserina has already yielded important information regarding the pathway regulating entrance into stationary phase. Here, the genes affected in two additional IDC mutants are identified as orthologues of the Saccharomyces cerevisiae WHI2 and PSR1 genes, known to regulate stationary phase in this yeast, arguing for a conserved role of these proteins throughout the evolution of ascomycetes.

International audience; Peroxisomes are versatile and dynamic organelles that are required for the development of diverse eukaryotic organisms. We demonstrated previously that in the fungus Podospora anserina different peroxisomal functions are required at distinct stages of sexual development, including the initiation and progression of meiocyte (ascus) development and the differentiation and germination of sexual spores (ascospores). Peroxisome assembly during these processes relies on the differential activity of the protein machinery that drives the import of proteins into the organelle, indicating a complex developmental regulation of peroxisome formation and activity. Here we demonstrate that peroxisome dynamics is also highly regulated during development. We show that peroxisomes in P. anserina are highly dynamic and respond to metabolic and environmental cues by undergoing changes in size, morphology and number. In addition, peroxisomes of vegetative and sexual cell types are structurally different. During sexual development peroxisome number increases at two stages: at early ascus differentiation and during ascospore formation. These processes are accompanied by changes in peroxisome structure and distribution, which include a cell-polarized concentration of peroxisomes at the beginning of ascus development, as well as a morphological transition from predominantly spherical to elongated shapes at the end of the first meiotic division. Further, the mostly tubular peroxisomes present from second meiotic division to early ascospore formation again become rounded during ascospore differentiation. Ultimately the number of peroxisomes dramatically decreases upon ascospore maturation. Our results reveal a precise regulation of peroxisome dynamics during sexual development and suggest that peroxisome constitution and function during development is defined by the coordinated regulation of the proteins that control peroxisome assembly and dynamics.

The endoplasmic reticulum (ER) is composed of distinct structural domains that perform diverse essential functions, including the synthesis of membrane lipids and proteins of the cell endomembrane system. The polarized growth of fungal hyphal cells depends on a polarized secretory system, which delivers vesicles to the hyphal apex for localized cell expansion, and that involves a polarized distribution of the secretory compartments, including the ER. Here we show that, additionally, the ER of the ascomycete Podospora anserina possesses a peripheral ER domain consisting of highly dynamic pleomorphic ER sub-compartments, which are specifically associated with the polarized growing apical hyphal cells.

Podospora anserinais a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation ofP. anserinato submerged cultivation, we initiated a long-term evolution experiment. In the course of the first four years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, the G-protein alpha subunit FadA evolved in seven out of eight experimental populations. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed allele replacements were adaptive and driven by positive selection.Author summaryLiving beings adapt to novel conditions that are far from their original environments in different ways. Studying mechanisms of adaptation is crucial for our understanding of evolution. The object of our interest is a multicellular fungusPodospora anserina. This fungus is known for its pronounced senescence and a definite lifespan, but it demonstrates an unlimited lifespan and no signs of senescence when grown under submerged conditions. Soon after transition to submerged cultivation, the rate of growth ofP. anserinaincreases and its pigmentation changes. We wanted to find out whether there are any genetic changes that contribute to adaptation ofP. anserinato these novel conditions and initiated a long-term evolutionary experiment on eight independent populations. Over the first four years of the experiment, 148 mutations were fixed in these populations. Many of these mutations lead to inactivation of the part of the developmental pathway inP. anserina, probably reallocating resources to vegetative proliferation in liquid medium. Our observations imply that strong positive selection drives changes in at least some of the affected protein-coding genes.Data AvailabilityGenome sequence data have been deposited at DDBJ/ENA/GenBank under accessions QHKV00000000 (founder genotype A; version QHKV01000000) and QHKU00000000 (founder genotype B; version QHKU01000000), with the respective BioSample accessions SAMN09270751 and SAMN09270757, under BioProject PRJNA473312. Sequencing data have been deposited at the SRA with accession numbers SRR7233712-SRR7233727, under the same BioProject.FundingExperimental work and sequencing were supported by the Russian Foundation for Basic Research (grants no. 16-04-01845a and 18-04-01349a). Bioinformatic analysis was supported by the Russian Science Foundation (grant no. 16-14-10173). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

As a late colonizer of herbivore dung, Podospora anserina has evolved an enzymatic machinery to degrade the more recalcitrant fraction of plant biomass, suggesting a great potential for biotechnology applications. The authors investigated its transcriptome during growth on two industrial feedstocks, soybean hulls (SBH) and corn stover (CS). Initially, CS and SBH results in the expression of hemicellulolytic and amylolytic genes, respectively, while at later time points a more diverse gene set is induced, especially for SBH. Substrate adaptation is also observed for carbon catabolism. Overall, SBH resulted in a larger diversity of expressed genes, confirming previous proteomics studies. The results not only provide an in depth view on the transcriptomic adaptation of P. anserina to substrate composition, but also point out strategies to improve saccharification of plant biomass at the industrial level.

WOS:000457456800015; International audience; Mechanisms involved in fine adaptation of fungi to their environment include differential gene regulation associated with single nucleotide polymorphisms and indels (including transposons), horizontal gene transfer, gene copy amplification, as well as pseudogenization and gene loss. The two Podospora genome sequences examined here emphasize the role of pseudogenization and gene loss, which have rarely been documented in fungi. Podospora comata is a species closely related to Podospora anserina, a fungus used as model in several laboratories. Comparison of the genome of P. comata with that of P. anserina, whose genome is available for over 10 years, should yield interesting data related to the modalities of genome evolution between these two closely related fungal species that thrive in the same types of biotopes, i.e., herbivore dung. Here, we present the genome sequence of the mat+isolate of the P. comata reference strain T. Comparison with the genome of the mat+isolate of P. anserina strain S confirms that P. anserina and P. comata are likely two different species that rarely interbreed in nature. Despite having a 94-99% of nucleotide identity in the syntenic regions of their genomes, the two species differ by nearly 10% of their gene contents. Comparison of the species-specific gene sets uncovered genes that could be responsible for the known physiological differences between the two species. Finally, we identified 428 and 811 pseudogenes (3.8 and 7.2% of the genes) in P. anserina and P. comata, respectively. Presence of high numbers of pseudogenes supports the notion that difference in gene contents is due to gene loss rather than horizontal gene transfers. We propose that the high frequency of pseudogenization leading to gene loss in P. anserina and P. comata accompanies specialization of these two fungi. Gene loss may be more prevalent during the evolution of other fungi than usually thought.