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Significance: Mitochondria are eukaryotic organelles with various essential functions. They are both the source and the targets of reactive oxygen species (ROS). Different branches of a mitochondrial quality control system (mQCS), such as ROS balancing, degradation of damaged proteins, or whole mitochondria, can mitigate the adverse effects of ROS stress. However, the capacity of mQCS is limited. Overwhelming this capacity leads to dysfunctions and aging. Strategies to interfere into mitochondria-dependent human aging with the aim to increase the healthy period of life, the health span, rely on the precise knowledge of mitochondrial functions. Experimental models such as Podospora anserina, a filamentous fungus with a clear mitochondrial aging etiology, proved to be instrumental to reach this goal. Recent Advances: Investigations of the P. anserina mQCS revealed that it is constituted by a complex network of different branches. Moreover, mitochondrial architecture and lipid homeostasis emerged to affect aging. Critical Issues: The regulation of the mQCS is only incompletely understood. Details about the involved signaling molecules and interacting pathways remain to be elucidated. Moreover, most of the currently generated experimental data were generated in well-controlled experiments that do not reflect the constantly changing natural life conditions and bear the danger to miss relevant aspects leading to incorrect conclusions. Future Directions: In P. anserina, the precise impact of redox signaling as well as of molecular damaging for aging remains to be defined. Moreover, natural fluctuation of environmental conditions needs to be considered to generate a realistic picture of aging mechanisms as they developed during evolution. [ABSTRACT FROM AUTHOR]

In filamentous fungi, vegetative cell fusion occurs within and between individuals. These fusions of growing hyphae (anastomosis) from two individuals produce binucleated cells with mixed cytoplasm known as heterokaryons. The fate of heterokaryotic cells was genetically controlled with delicacy by specific loci named het (heterokaryon) or vic (vegetative incompatibility) as a part of self-/nonself-recognition system. When het loci of two individuals are incompatible, the resulting heterokaryotic cells underwent programmed cell death or showed severely impaired fungal growth. In Podospora anserina, het-s is one of at least nine alleles that control heterokaryon incompatibility and the altered protein conformation [Het-s] prion. The present study describes the [Het-s] prion in terms of (1) the historical discovery based on early genetic and physiological studies, (2) architecture built on its common and unique nature compared with other prions, and (3) functions related to meiotic drive and programmed cell death. [ABSTRACT FROM AUTHOR]

AbstractIn filamentous fungi, vegetative cell fusion occurs within and between individuals. These fusions of growing hyphae (anastomosis) from two individuals produce binucleated cells with mixed cytoplasm known as heterokaryons. The fate of heterokaryotic cells was genetically controlled with delicacy by specific loci named het (heterokaryon) or vic (vegetative incompatibility) as a part of self-/nonself-recognition system. When het loci of two individuals are incompatible, the resulting heterokaryotic cells underwent programmed cell death or showed severely impaired fungal growth. In Podospora anserina, het-s is one of at least nine alleles that control heterokaryon incompatibility and the altered protein conformation [Het-s] prion. The present study describes the [Het-s] prion in terms of (1) the historical discovery based on early genetic and physiological studies, (2) architecture built on its common and unique nature compared with other prions, and (3) functions related to meiotic drive and programmed cell death.

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina , high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification. [ABSTRACT FROM AUTHOR]

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species. Author summary: Many cellular life forms display genetic systems that protect individuality and discriminate conspecific self from non-self. In filamentous fungi, cell fusion events between strains are under check by specific allorecognition genes that trigger regulated cell death upon detection of non-self. The role of incompatibility is to restrict mycovirus transmission and conspecific parasitism. Podospora anserina, a good model for the study of this form of allorecognition, harbors nine incompatibility het loci. Previous studies have revealed that these genes can be homologous to genes with immune functions in other phyla including bacteria, plants and animals. We have cloned het-B, the last of the nine het genes that remained to be identified and found that it is a complex locus comprising two adjacent genes Bh and Bp. BH displays an N-terminal HET domain (related to TIR domains) and a C-terminal domain with a predicted lectin fold. BP is homologous to PII-like proteins, known bacterial metabolite sensors. Intriguingly, despite apparent genetic allelism, incompatibility is dictated by the non-allelic Bh/Bp interaction. This study stresses the reoccurring involvement of HET domains in fungal RCD and signs completion of the characterization of the entire set of het loci in that species, enabling a comparative analysis of the different genetic architectures underlying allorecognition. [ABSTRACT FROM AUTHOR]

In their natural environment, fungi are subjected to a wide variety of environmental stresses which they must cope with by constantly adapting the architecture of their growing network. In this work, our objective was to finely characterize the thallus development of the filamentous fungus Podospora anserina subjected to different constraints that are simple to implement in vitro and that can be considered as relevant environmental stresses, such as a nutrient-poor environment or non-optimal temperatures. At the Petri dish scale, the observations showed that the fungal thallus is differentially affected (thallus diameter, mycelium aspect) according to the stresses but these observations remain qualitative. At the hyphal scale, we showed that the extraction of the usual quantities (i.e. apex, node, length) does not allow to distinguish the different thallus under stress, these quantities being globally affected by the application of a stress in comparison with a thallus having grown under optimal conditions. Thanks to an original geomatics-based approach based on the use of automatized Geographic Information System (GIS) tools, we were able to produce maps and metrics characterizing the growth dynamics of the networks and then to highlight some very different dynamics of network densification according to the applied stresses. The fungal thallus is then considered as a map and we are no longer interested in the quantity of material (hyphae) produced but in the empty spaces between the hyphae, the intra-thallus surfaces. This study contributes to a better understanding of how filamentous fungi adapt the growth and densification of their network to potentially adverse environmental changes. [ABSTRACT FROM AUTHOR]

Background: The genome of the filamentous ascomycete Podospora anserina shows a relatively high abundance of retrotransposons compared to other interspersed repeats. The LTR-retrotransposon family crapaud is particularly abundant in the genome, and consists of multiple diverged sequence variations specifically localized in the 5' half of both long terminal repeats (LTRs). P. anserina is part of a recently diverged species-complex, which makes the system ideal to classify the crapaud family based on the observed LTR variation and to study the evolutionary dynamics, such as the diversification and bursts of the elements over recent evolutionary time. Results: We developed a sequence similarity network approach to classify the crapaud repeats of seven genomes representing the P. anserina species complex into 14 subfamilies. This method does not utilize a consensus sequence, but instead it connects any copies that share enough sequence similarity over a set sequence coverage. Based on phylogenetic analyses, we found that the crapaud repeats likely diversified in the ancestor of the complex and have had activity at different time points for different subfamilies. Furthermore, while we hypothesized that the evolution into multiple subfamilies could have been a direct effect of escaping the genome defense system of repeat induced point mutations, we found this not to be the case. Conclusions: Our study contributes to the development of methods to classify transposable elements in fungi, and also highlights the intricate patterns of retrotransposon evolution over short timescales and under high mutational load caused by nucleotide-altering genome defense. [ABSTRACT FROM AUTHOR]

The ascomycete Podospora anserina is a heterothallic filamentous fungus found mainly on herbivore dung. It is commonly used in laboratories as a model system, and its complete life cycle lasting eight days is well mastered in vitro. The main objective of our team is to understand better the global process of fruiting body development, named perithecia, induced normally in this species by fertilization. Three allelic mutants, named pfd3, pfd9, and pfd23 (for "promoting fruiting body development") obtained by UV mutagenesis, were selected in view of their abilities to promote barren perithecium development without fertilization. By complete genome sequencing of pfd3 and pfd9, and mutant complementation, we identified point mutations in the mcm1 gene as responsible for spontaneous perithecium development. MCM1 proteins are MADS box transcription factors that control diverse developmental processes in plants, metazoans, and fungi. We also identified using the same methods a mutation in the VelC gene as responsible for spontaneous perithecium development in the vacua mutant. The VelC protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production. A key role of MCM1 and VelC in coordinating the development of P. anserina perithecia with gamete formation and fertilization is highlighted. [ABSTRACT FROM AUTHOR]

Biomembranes fulfill several essential functions. They delimitate cells and control the exchange of compounds between cells and the environment. They generate specialized cellular reaction spaces, house functional units such as the respiratory chain (RC), and are involved in content trafficking. Biomembranes are dynamic and able to adjust their properties to changing conditions and requirements. An example is the inner mitochondrial membrane (IMM), which houses the RC involved in the formation of adenosine triphosphate (ATP) and the superoxide anion as a reactive oxygen species (ROS). The IMM forms a characteristic ultrastructure that can adapt to changing physiological situations. In the fungal aging model Podospora anserina, characteristic age-related changes of the mitochondrial ultrastructure occur. More recently, the impact of membranes on aging was extended to membranes involved in autophagy, an important pathway involved in cellular quality control (QC). Moreover, the effect of oleic acid on the lifespan was linked to basic biochemical processes and the function of membranes, providing perspectives for the elucidation of the mechanistic effects of this nutritional component, which positively affects human health and aging.

The Podospora anserina long-term evolution experiment (PaLTEE) is the only running filamentous fungus study, which is still going on. The aim of our work is to trace the evolutionary dynamics of the accumulation of mutations in the genomes of eight haploid populations of P. anserina. The results of the genome-wide analysis of all of the lineages, performed 8 years after the start of the PaLTEE, are presented. Data analysis detected 312 single nucleotide polymorphisms (SNPs) and 39 short insertion-deletion mutations (indels) in total. There was a clear trend towards a linear increase in the number of SNPs depending on the experiment duration. Among 312 SNPs, 153 were fixed in the coding regions of P. anserina genome. Relatively few synonymous mutations were found, exactly 38; 42 were classified as nonsense mutations; 72 were assigned to missense mutations. In addition, 21 out of 39 indels identified were also localized in coding regions. Here, we also report the detection of parallel evolution at the paralog level in the P. anserina model system. Parallelism in evolution at the level of protein functions also occurs. The latter is especially true for various transcription factors, which may indicate selection leading to optimization of the wide range of cellular processes under experimental conditions. [ABSTRACT FROM AUTHOR]

Under ideal conditions, the growth of the mycelial network of a filamentous fungus is monotonous, showing an ever increasing complexity with time. The components of the network growth are very simple and based on two mechanisms: the elongation of each hypha, and their multiplication by successive branching. These two mechanisms are sufficient to produce a complex network, and could be localized only at the tips of hyphae. However, branching can be of two types, apical or lateral, depending on its location on the hyphae, therefore imposing the redistribution of the necessary material in the whole mycelium. From an evolutionary point of view, maintaining different branching processes, with additional energy needs for structure and metabolism, is intriguing. We propose in this work to discuss the advantages of each branching type using a new observable for the network growth, allowing us to compare growth configurations. For this purpose, we build on experimental observations of the Podospora anserina mycelium growth, enabling us to feed and constrain a lattice-free modeling of this network based on a binary tree. First, we report the set of statistics related to the branches of P. anserina that we have implemented into the model. Then, we build the density observable, allowing us to discuss the succession of growth phases. We predict that density over time is not monotonic, but shows a decay growth phase, clearly separated from an other one by a stationary phase. The time of appearance of this stable region appears to be driven solely by the growth rate. Finally, we show that density is an appropriate observable to differentiate growth stress. [ABSTRACT FROM AUTHOR]

The ascomycete Podospora anserina is a heterothallic filamentous fungus found mainly on herbivore dung. It is commonly used in laboratories as a model system, and its complete life cycle lasting eight days is well mastered in vitro. The main objective of our team is to understand better the global process of fruiting body development, named perithecia, induced normally in this species by fertilization. Three allelic mutants, named pfd3, pfd9, and pfd23 (for “promoting fruiting body development”) obtained by UV mutagenesis, were selected in view of their abilities to promote barren perithecium development without fertilization. By complete genome sequencing of pfd3 and pfd9, and mutant complementation, we identified point mutations in the mcm1 gene as responsible for spontaneous perithecium development. MCM1 proteins are MADS box transcription factors that control diverse developmental processes in plants, metazoans, and fungi. We also identified using the same methods a mutation in the VelC gene as responsible for spontaneous perithecium development in the vacua mutant. The VelC protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production. A key role of MCM1 and VelC in coordinating the development of P. anserina perithecia with gamete formation and fertilization is highlighted.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression. Author summary: Recombination allows faster adaptation and the purging of deleterious mutations but is often paradoxically lacking in sex chromosomes. It has been recently recognized that recombination can also be suppressed on fungal mating-type chromosomes, but the evolutionary explanation and the proximal mechanism of this phenomenon remain unclear. By studying here the sexual biology of a poorly studied mold living in rabbit dung, we reveal a striking convergence in three distant fungal lineages of an independently evolved association between the production of self-fertile sexual spores (carrying two nuclei with opposite mating types), a particular segregation of the mating-type locus and stepwise recombination suppression on mating-type chromosomes. Such a convergent association suggests causal relationships and will contribute to unveil the evolutionary causes of recombination suppression. [ABSTRACT FROM AUTHOR]

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment. [ABSTRACT FROM AUTHOR]

Fungi have a large potential for flexibility in their mode of sexual reproduction, resulting in mating systems ranging from haploid selfing to outcrossing. However, we know little about which mating strategies are used in nature, and why, even in well‐studied model organisms. Here, we explored the fitness consequences of alternative mating strategies in the ascomycete fungus Podospora anserina. We measured and compared fitness proxies of nine genotypes in either diploid selfing or outcrossing events, over two generations, and with or without environmental stress. We showed that fitness was consistently lower in outcrossing events, irrespective of the environment. The cost of outcrossing was partly attributed to non‐self recognition genes with pleiotropic effects on fertility. We then predicted that when presented with options to either self or outcross, individuals would perform mate choice in favour of the reproductive strategy that yields higher fitness. Contrary to our prediction, individuals did not seem to avoid outcrossing when a choice was offered, in spite of the fitness cost incurred. Our results suggest that, although functionally diploid, P. anserina does not benefit from outcrossing in most cases. We outline different explanations for the apparent lack of mate choice in face of high fitness costs associated with outcrossing, including a new perspective on the pleiotropic effect of non‐self recognition genes. [ABSTRACT FROM AUTHOR]

Background: Publicly available RNA-seq datasets are often underused although being helpful to improve functional annotation of eukaryotic genomes. This is especially true for filamentous fungi genomes which structure differs from most well annotated yeast genomes. Podospora anserina is a filamentous fungal model, which genome has been sequenced and annotated in 2008. Still, the current annotation lacks information about cis-regulatory elements, including promoters, transcription starting sites and terminators, which are instrumental to integrate epigenomic features into global gene regulation strategies. Results: Here we took advantage of 37 RNA-seq experiments that were obtained in contrasted developmental and physiological conditions, to complete the functional annotation of P. anserina genome. Out of the 10,800 previously annotated genes, 5'UTR and 3'UTR were defined for 7554, among which, 3328 showed differential transcriptional signal starts and/or transcriptional end sites. In addition, alternative splicing events were detected for 2350 genes, mostly due alternative 3'splice sites and 1732 novel transcriptionally active regions (nTARs) in unannotated regions were identified. Conclusions: Our study provides a comprehensive genome-wide functional annotation of P. anserina genome, including chromatin features, cis-acting elements such as UTRs, alternative splicing events and transcription of non-coding regions. These new findings will likely improve our understanding of gene regulation strategies in compact genomes, such as those of filamentous fungi. Characterization of alternative transcripts and nTARs paves the way to the discovery of putative new genes, alternative peptides or regulatory non-coding RNAs. [ABSTRACT FROM AUTHOR]

Sexual reproduction in Ascomycetes is well described in several model organisms such as Neurospora crassa or Podospora anserina. Deciphering the biological process of sexual reproduction (from the recognition between compatible partners to the formation of zygote) can be a major advantage to better control sexually reproducing pathogenic fungi. In Pyricularia oryzae, the fungal pathogen causing blast diseases on several Poaceae species, the biology of sexual reproduction remains poorly documented. Besides the well‐documented production of asexual macroconidia, the production of microconidia was seldom reported in P. oryzae, and their role as male gamete (i.e., spermatia) and in male fertility has never been explored. Here, we characterised the morphological features of microconidia and demonstrated that they are bona fide spermatia. Contrary to macroconidia, microconidia are not able to germinate and seem to be the only male gametes in P. oryzae. We show that fruiting body (perithecium) formation requires microconidia to get in contact with mycelium of strains of opposite mating type, to presumably fertilise the female gametes. [ABSTRACT FROM AUTHOR]

Based upon apical growth and hyphal branching, the two main processes that drive the growth pattern of a fungal network, we propose here a two-dimensions simulation based on a binary-tree modelling allowing us to extract the main characteristics of a generic thallus growth. In particular, we showed that, in a homogeneous environment, the fungal growth can be optimized for exploration and exploitation of its surroundings with a specific angular distribution of apical branching. Two complementary methods of extracting angle values have been used to confront the result of the simulation with experimental data obtained from the thallus growth of the saprophytic filamentous fungus Podospora anserina. Finally, we propose here a validated model that, while being computationally low-cost, is powerful enough to test quickly multiple conditions and constraints. It will allow in future works to deepen the characterization of the growth dynamic of fungal network, in addition to laboratory experiments, that could be sometimes expensive, tedious or of limited scope. [ABSTRACT FROM AUTHOR]

Summary: Light serves as a source of information and regulates diverse physiological processes in living organisms. Fungi perceive and respond to light through a complex photosensory system. Fungi have evolved the desensitization mechanism to adapt to the changing light signal in a natural environment. White light exerts multiple essential impacts on the model filamentous fungus Podospora anserina. However, the light sensing and response in this species has not been investigated. In this study, we demonstrated that the loss of function of the light desensitization protein VIVID (VVD) in P. anserina triggered exacerbated light responses and therefore led to drastic morphological and physiological changes. The white light‐sensitive mutant Δvvd showed growth reduction, spermatia overproduction, enhanced hyphae pigmentation and reduced oxidative stress tolerance. We observed the decreased expression level of sterigmatocystin gene cluster by transcriptome analysis and finally detected the reduced production of sterigmatocystin in Δvvd in response to white light. Our data indicate that VVD acts as a repressor of white collar complex. This study exhibits a vital role of VVD in governing white light‐responsive gene expression and secondary metabolite production and contributes to a better understanding of the photoreceptor VVD in P. anserina. [ABSTRACT FROM AUTHOR]

Abstract 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.

Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4- O -methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-β-pNP) and coupled with the activity of GH67 (α-glucuronidase) and GH43 (β-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-β-pNP) were proteins originating from Schizophyllum commune, Podospora anserina , Trametes versicolor , and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations. • Expression and activity screening of 15 putative glucuronoyl esterases. • Identification of 7 novel enzymes that display glucuronoyl esterase activity. • Implementation of a simple screening strategy, which mimics natural lignin-carbohydrate complexes. [ABSTRACT FROM AUTHOR]

10.1007/978-3-319-27790-5 7 14 Lim J.-Y., Kim Y.-J., Park H.-M. The G -like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus. Similarly, the interplay between two signaling pathways was studied by Moreno-Ruiz et al. [[20]] They investigated the stress-activated protein kinase signaling (SAPK) and mitogen-activated protein kinase (MAPK) signaling network in I Trichoderma atroviride i , a mycoparasitic fungus. Bühring et al. [[6]] investigated the phosphotransferase system in the rice blast fungus I Magnaporthe oryzae i and addressed the question of how a single gene for a phosphotransfer protein may generate several isoforms to contribute to a multistep phospho-relay system. A deletion strain lacking the G protein subunit gene shows significantly reduced transcription of genes involved in cAMP signaling. [Extracted from the article]

Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the 'mitochondrial contact site and cristae organizing system' (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina. [ABSTRACT FROM AUTHOR]

The model ascomycete Podospora anserina, distinguished by its strict sexual development, is a prolific but yet unexploited reservoir of natural products. The GATA-type transcription factor NsdD has been characterized by the role in balancing asexual and sexual reproduction and governing secondary metabolism in filamentous fungi. In the present study, we functionally investigated the NsdD ortholog PaNsdD in P. anserina. Compared to the wild-type strain, vegetative growth, ageing processes, sexual reproduction, stress tolerance, and interspecific confrontations in the mutant were drastically impaired, owing to the loss of function of PaNsdD. In addition, the production of 3-acetyl-4-methylpyrrole, a new metabolite identified in P. anserina in this study, was significantly inhibited in the DPaNsdD mutant. We also demonstrated the interplay of PaNsdD with the sterigmatocystin biosynthetic gene pathway, especially as the deletion of PaNsdD triggered the enhanced red-pink pigment biosynthesis that occurs only in the presence of the core polyketide synthase-encoding gene PaStcA of the sterigmatocystin pathway. Taken together, these results contribute to a better understanding of the global regulation mediated by PaNsdD in P. anserina, especially with regard to its unexpected involvement in the fungal ageing process and its interplay with the sterigmatocystin pathway. [ABSTRACT FROM AUTHOR]

Vegetative incompatibility (VI) is a widespread phenomenon developed in Morchella importuna , a species of ascomycete fungus that is cultivated on a rapidly expanding scale in China. Understanding the genetic bases of this nonself-recognition phenomenon is beneficial for resolving some problems that are associated with the production of this highly prized edible fungus, such as crossbreeding, strain classification, and pathogen transmission. VI is genetically controlled by het genes, organized in two different systems, namely allelic and nonallelic. These het genes have been well characterized in Podospora anserina and Neurospora crassa. In this work, putative het -homologs were identified in the genome of M. importuna , but their low allelic polymorphism in different vegetative compatibility groups (VCGs) suggested that VI in this fungus might not be regulated by these het genes. The progeny derived from vegetative compatible parents became a VCG, while the single-ascospore strains from vegetative incompatible parents were divided into four VCGs, and the interaction between the inter-group strains led to the formation of two types of barrages, viz., thin dark line and raised aggregate of hyphae. The Bulk Segregant Analysis confirmed that the genes mimpvic32 and mimpvic33 were linked to VI reactions in M. importuna ; nevertheless, the formation of barrages also occurred between the pairs carrying the same allele of these two genes. In sum, the VI control system in M. importuna was complicated, and there were more other allelic or non-allelic VI-related genes. [ABSTRACT FROM AUTHOR]

Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death-inducing toxins in the context of conspecific non-self-discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina. We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a ∼5-kDa C-terminal fragment during the cell death reaction in the presence of a subtilisin-like serine protease termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in heterologous hosts (Saccharomyces cerevisiae and human 293T cells) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analyzing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2-like proteins carry either subtilisin-like or caspase-related proteases, which, in some cases, correspond to the N-terminal effector domain of nucleotide-binding and oligomerization-like receptor proteins. This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • An efficient method for the analysis of protein carbonylation in fungi has been developed. • Revealed four main features of the accumulation of carbonylated proteins in fungi. • Two polar strategies for the accumulation of carbonylated proteins in different fungal species are described. [ABSTRACT FROM AUTHOR]

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

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. [ABSTRACT FROM AUTHOR]

In nature, it is often surmised that lignin is degraded almost exclusively by basidiomycetes, with minor contributions from bacteria and even fewer from ascomycetes. Yet, numerous studies underscore the ability of many ascomycetes from a wide range of taxonomic classes to degrade newly-formed and ancient lignin, sometime more efficiently than basidiomycetes. Few genomes from ascomycetes have been analyzed in relation with their potential ability to breakdown lignin; yet, these also point out a rich repertoire of enzymes with auxiliary activities suspected to participate in lignin lysis. Not surprisingly, the molecular mechanisms used by ascomycetes to break down lignin are largely unknown. Here, we review the literature on this topic. We also present potential mechanisms by which Podospora anserina, a model ascomycete well-suited to decipher how ascomycetes breakdown lignin, may do so. Owing to their huge biomass, which dominates in soils, and despite the fact that they usually are less efficient than basidiomycetes, it is most likely that ascomycetes actually play important roles in ensuring the recycling of lignin in nature. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the agerelated reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated. [ABSTRACT FROM AUTHOR]

Organismic aging is known to be controlled by genetic and environmental traits. Pathways involved in the control of cellular metabolism play a crucial role. Previously, we identified a role of PaCLPP, a mitochondrial matrix protease, in the control of the mitochondrial energy metabolism, aging, and lifespan of the fungal aging model Podospora anserina. Most surprisingly, we made the counterintuitive observation that the ablation of this component of the mitochondrial quality control network leads to lifespan extension. In the current study, we investigated the role of energy metabolism of P. anserina. An age-dependent metabolome analysis of the wild type and a PaClpP deletion strain verified differences and changes of various metabolites in cultures of the PaClpP mutant and the wild type. Based on these data, we generated and analyzed a PaSnf1 deletion mutant and a ΔPaSnf1/ΔPaClpP double mutant. In both mutants PaSNF1, the catalytic α-subunit of AMP-activated protein kinase (AMPK) is ablated. PaSNF1 was found to be required for the development of fruiting bodies and ascospores and the progeny of sexual reproduction of this ascomycete and impact mitochondrial dynamics and autophagy. Most interestingly, while the single PaSnf1 deletion mutant is characterized by a slight lifespan increase, simultaneous deletion of PaSnf1 and PaClpP leads to a pronounced lifespan extension. This synergistic effect is strongly reinforced in the presence of the mating-type “minus”-linked allele of the rmp1 gene. Compared to the wild type, culture temperature of 35°C instead of the standard laboratory temperature of 27°C leads to a short-lived phenotype of the ΔPaSnf1/ΔPaClpP double mutant. Overall, our study provides novel evidence for complex interactions of different molecular pathways involved in mitochondrial quality control, gene expression, and energy metabolism in the control of organismic aging.

Summary: Genomic regions determining sexual compatibility often display recombination suppression, as occurs in sex chromosomes, plant self‐incompatibility loci and fungal mating‐type loci. Regions lacking recombination can extend beyond the genes determining sexes or mating types, by several successive steps of recombination suppression. Here we review the evidence for recombination suppression around mating‐type loci in fungi, sometimes encompassing vast regions of the mating‐type chromosomes. The suppression of recombination at mating‐type loci in fungi has long been recognized and maintains the multiallelic combinations required for correct compatibility determination. We review more recent evidence for expansions of recombination suppression beyond mating‐type genes in fungi ('evolutionary strata'), which have been little studied and may be more pervasive than commonly thought. We discuss testable hypotheses for the ultimate (evolutionary) and proximate (mechanistic) causes for such expansions of recombination suppression, including (1) antagonistic selection, (2) association of additional functions to mating‐type, such as uniparental mitochondria inheritance, (3) accumulation in the margin of nonrecombining regions of various factors, including deleterious mutations or transposable elements resulting from relaxed selection, or neutral rearrangements resulting from genetic drift. The study of recombination suppression in fungi could thus contribute to our understanding of recombination suppression expansion across a broader range of organisms. [ABSTRACT FROM AUTHOR]

Summary: Secreted proteins are key players in fungal physiology and cell protection against external stressing agents and antifungals. Oak stress‐induced protein 1 (OSIP1) is a fungal‐specific protein with unknown function. By using Podospora anserina and Phanerochaete chrysosporium as models, we combined both in vivo functional approaches and biophysical characterization of OSIP1 recombinant protein. The P. anserina OSIP1Δ mutant showed an increased sensitivity to the antifungal caspofungin compared to the wild type. This correlated with the production of a weakened extracellular exopolysaccharide/protein matrix (ECM). Since the recombinant OSIP1 from P. chrysosporium self‐assembled as fibers and was capable of gelation, it is likely that OSIP1 is linked to ECM formation that acts as a physical barrier preventing drug toxicity. Moreover, compared to the wild type, the OSIP1Δ mutant was more sensitive to oak extractives including chaotropic phenols and benzenes. It exhibited a strongly modified secretome pattern and an increased production of proteins associated to the cell‐wall integrity signalling pathway, when grown on oak sawdust. This demonstrates that OSIP1 has also an important role in fungal resistance to extractive‐induced stress. [ABSTRACT FROM AUTHOR]

Neurodegenerative disorders are frequently associated with ß-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical ß-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents. [ABSTRACT FROM AUTHOR]

The sucrose non-fermenting 1/AMP-activated protein kinase (SNF1/AMPK) is a central regulator of carbon metabolism and energy production in the eukaryotes. In this study, the functions of the Podospora anserina SNF1 (PaSNF1) ortholog were investigated. The ΔPaSNF1 mutant displays a delayed development of mycelium and fruiting bodies and fails to form ascospores. The expression of the PaSNF1 gene in the strain providing female organs in a cross is sufficient to ensure fertility, indicating a maternal effect. Results of environmental stress showed that ΔPaSNF1 was hypersensitive to stress, such as osmotic pressure and heat shock, and resistant to fluconazole. Interestingly, the knockout of PaSNF1 significantly promoted sterigmatocystin (ST) synthesis but suppressed cellulase [filter paperase (FPA), endoglucanase (EG), and β-glucosidase (BG)] activity. Further, transcriptome analysis indicated that PaSNF1 made positive regulatory effects on the expression of genes encoding cellulolytic enzymes. These results suggested that PaSNF1 may function in balancing the operation of primary and secondary metabolism. This study suggested that SNF1 was a key regulator concerting vegetative growth, sexual development, and stress tolerance. Our study provided the first genetic evidence that SNF1 was involved in the ST biosynthesis and that it may also be a major actor of lignocellulose degradation in P. anserina.

The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.

Various components in the cell are responsible for maintaining physiological levels of reactive oxygen species (ROS). Several different enzymes exist that can convert or degrade ROS; among them are the superoxide dismutases (SODs). If left unchecked, ROS can cause damage that leads to pathology, can contribute to aging, and may, ultimately, cause death. SODs are responsible for converting superoxide anions to hydrogen peroxide by dismutation. Here we review the role of different SODs on the development and pathogenicity of various eukaryotic microorganisms relevant to human health. These include the fungal aging model, Podospora anserina; various members of the genus Aspergillus that can potentially cause aspergillosis; the agents of diseases such as Chagas and sleeping disease, Trypanosoma cruzi and Trypanosoma brucei, respectively; and, finally, pathogenic amoebae, such as Acanthamoeba spp. In these organisms, SODs fulfill essential and often regulatory functions that come into play during processes such as the development, host infection, propagation, and control of gene expression. We explore the contribution of SODs and their related factors in these microorganisms, which have an established role in health and disease.

The filamentous fungus Podospora anserina is a good model to study the breakdown of lignocellulose, owing to its ease of culture and genetical analysis. Here, we show that the fungus is able to use a wide range of lignocellulosic materials as food sources. Using color assays, spectroscopy and pyrolysis-gas chromatography mass spectrometry, we confirm that this ascomycete is able to degrade lignin, primarily by hydrolyzing β-O-4 linkages, which facilitates its nutrient uptake. We show that the limited weight loss that is promoted when attacking Miscanthus giganteus is due to a developmental blockage rather than an inefficiency of its enzymes. Finally, we show that lignin, and, more generally, phenolics, including degradation products of lignin, greatly stimulate the growth and fertility of the fungus in liquid cultures. Analyses of the CATDDDDD mutant lacking all its catalases, pro-oxidants and antioxidants indicate that improved growth and fertility of the fungus is likely caused by augmented reactive oxygen species levels triggered by the presence of phenolics. [ABSTRACT FROM AUTHOR]

The sucrose non-fermenting 1/AMP-activated protein kinase (SNF1/AMPK) is a central regulator of carbon metabolism and energy production in the eukaryotes. In this study, the functions of the Podospora anserina SNF1 (PaSNF1) ortholog were investigated. The Δ PaSNF1 mutant displays a delayed development of mycelium and fruiting bodies and fails to form ascospores. The expression of the PaSNF1 gene in the strain providing female organs in a cross is sufficient to ensure fertility, indicating a maternal effect. Results of environmental stress showed that Δ PaSNF1 was hypersensitive to stress, such as osmotic pressure and heat shock, and resistant to fluconazole. Interestingly, the knockout of PaSNF1 significantly promoted sterigmatocystin (ST) synthesis but suppressed cellulase [filter paperase (FPA), endoglucanase (EG), and β-glucosidase (BG)] activity. Further, transcriptome analysis indicated that PaSNF1 made positive regulatory effects on the expression of genes encoding cellulolytic enzymes. These results suggested that PaSNF1 may function in balancing the operation of primary and secondary metabolism. This study suggested that SNF1 was a key regulator concerting vegetative growth, sexual development, and stress tolerance. Our study provided the first genetic evidence that SNF1 was involved in the ST biosynthesis and that it may also be a major actor of lignocellulose degradation in P. anserina. [ABSTRACT FROM AUTHOR]

Background: The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results: Quantitative 13C lignin internal standard py-GC–MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC–MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions: For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms. [ABSTRACT FROM AUTHOR]

Mitochondria and peroxisomes are organelles whose activity is intimately associated and that play fundamental roles in development. In the model fungus Podospora anserina , peroxisomes and mitochondria are required for different stages of sexual development, and evidence indicates that their activity in this process is interrelated. Additionally, sexual development involves precise regulation of peroxisome assembly and dynamics. Peroxisomes and mitochondria share the proteins mediating their division. The dynamin-related protein Dnm1 (Drp1) along with its membrane receptors, like Fis1, drives this process. Here we demonstrate that peroxisome and mitochondrial fission in P. anserina depends on FIS1 and DNM1. We show that FIS1 and DNM1 elimination affects the dynamics of both organelles throughout sexual development in a developmental stage-dependent manner. Moreover, we discovered that the segregation of peroxisomes, but not mitochondria, is affected upon elimination of FIS1 or DNM1 during the division of somatic hyphae and at two central stages of sexual development—the differentiation of meiocytes (asci) and of meiotic-derived spores (ascospores). Furthermore, we found that FIS1 and DNM1 elimination results in delayed karyogamy and defective ascospore differentiation. Our findings reveal that sexual development relies on complex remodeling of peroxisomes and mitochondria, which is driven by their common fission machinery. [ABSTRACT FROM AUTHOR]

The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.

Abstract 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.

Biomembranes fulfill several essential functions. They delimitate cells and control the exchange of compounds between cells and the environment. They generate specialized cellular reaction spaces, house functional units such as the respiratory chain (RC), and are involved in content trafficking. Biomembranes are dynamic and able to adjust their properties to changing conditions and requirements. An example is the inner mitochondrial membrane (IMM), which houses the RC involved in the formation of adenosine triphosphate (ATP) and the superoxide anion as a reactive oxygen species (ROS). The IMM forms a characteristic ultrastructure that can adapt to changing physiological situations. In the fungal aging model Podospora anserina , characteristic age-related changes of the mitochondrial ultrastructure occur. More recently, the impact of membranes on aging was extended to membranes involved in autophagy, an important pathway involved in cellular quality control (QC). Moreover, the effect of oleic acid on the lifespan was linked to basic biochemical processes and the function of membranes, providing perspectives for the elucidation of the mechanistic effects of this nutritional component, which positively affects human health and aging., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author declares being an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Osiewacz.)

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. [ABSTRACT FROM AUTHOR]

• Inactivation of the phosphatase domain of PFK2/FBPase2 suppresses the Δcox phenotype. • The suppressor effect results from the activation of the RSEs transcription factors. • Suppression of the Δcox phenotype results from the alternative oxidase expression. • Killing the phosphatase domain of PFK-2/FBPase-2 induces the gluconeogenic enzymes. • Multiple PFK-2/FBPase-2 isoforms are present in all the species of tested Ascomycetes. 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. [ABSTRACT FROM AUTHOR]