The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.     

Purification and characterization of an endo-exonuclease from Podospora anserina mitochondria

The senescence phenotype of Podospora anserina wild-type strains depends on mitochondrial (mt) genome stability. Characterization of activities implicated in the maintenance of the mt DNA is therefore essential for a better understanding of these degenerative processes. To address this question we looked for a nuclease activity in this fungal mitochondria. Here we describe the purification of an endo-exonuclease active on single-stranded, double-stranded and flap DNA. The Podospora nuclease also possesses an RNase H activity. Gel filtration chromatography showed a native molecular mass of 90 kDa for the P. anserina enzyme. The highly purified fraction shows a single polypeptide chain of 49 kDa on SDS-PAGE, indicating that the Podospora enzyme is probably active as a dimer. Purification and sequencing of the endolysine digestion peptides of the Podospora mt nuclease suggested that this enzyme could belong to the 5' structure-specific endo-exonuclease family. The possible involvement of this nuclease in mt DNA recombination during the senescence process is evoked.     

Accelerated cell death in Podospora autophagy mutants

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The DeltaPaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent protein-PaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in DeltaPaATG1 and DeltaPaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in DeltaPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.     

Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity

Prions have been described in mammals and fungi. The [Het-s] infectious genetic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. This protein is involved in the control of a cell death reaction termed heterokaryon incompatibility. The infectious form of HET-s corresponds to a self-perpetuating amyloid. The purpose of the present paper is to describe the techniques that can be used to analyse [Het-s] prion propagation in vivo and HET-s amyloid aggregation in vitro. In addition, we report several methods that can be used to infect Podospora with recombinant HET-s amyloid.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Aggregation of amyloid proteins is involved in serious neurodegenerative disorders such as Alzheimer disease and transmissible encephalopathies. The concept of an infectious protein (prion) proposed as the scrapie agent was successfully validated for several proteins of yeast and fungi. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically, then biochemically identified as prion proteins. Studies on these proteins have brought critical informations on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by a rich β-sheet content. In a previous work on the HET-s prion protein of Podospora we have demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the prion domain of HET-s associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review as well as relevant questions about the [Het-s] system of Podospora anserina.     

Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.     

Probing eukaryotic RNA polymerases B with monoclonal antibodies

Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora comata were selected on the basis of different subunits recognition and inhibitory effect on enzyme activity. A library of 10 antibodies biased toward B180, B145, B39, B23,5 and B11 subunits was constructed. Most of these antibodies also recognize yeast, wheat germ and calf thymus RNA polymerase B. Subunits bearing antigenic determinants are not always homologous in Podospora and yeast enzyme. As some of these antibodies strongly inhibit enzyme activity they constitute potent probes for functional studies of corresponding subunits.     

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.     

The mitochondrial plasmid pAL2-1 reduces calorie restriction mediated life span extension in the filamentous fungus Podospora anserina

Calorie restriction is the only life span extending regimen known that applies to all aging organisms. Although most fungi do not appear to senesce, all natural isolates of the modular filamentous fungus Podospora anserina have a limited life span. In this paper, we show that calorie restriction extends life span also in Podospora anserina. The response to glucose limitation varies significantly among 23 natural isolates from a local population in The Netherlands, ranging from no effect up to a 5-fold life span extension. The isolate dependent effect is largely due to the presence or absence of pAL2-1 homologous plasmids. These mitochondrial plasmids are associated with reduced life span under calorie restricted conditions, suggesting a causal link. This has been substantiated using three combinations of isogenic isolates with and without plasmids. A model is proposed to explain how pAL2-1 homologues influence the response to calorie restriction.     

A Short History of Small s: A Prion of the Fungus Podospora anserina

Prions are infectious proteins. In fungi, prions correspond to non-Mendelian genetic elements whose mode of inheritance has long eluded explanation. The [Het-s] cytoplasmic genetic element of the filamentous fungus Podospora anserina, was originally identified in 1952 and recognized as a prion nearly half a century later. The present chapter will attempt to describe the work on [Het-s] from a historical perspective. The initial characterization and early genetic and physiological studies of [Het-s] are described together with the isolation of the [Het-s] encoding gene. More recent work that led to the construction of a structural model for this prion is also discussed.Key Words: prion, Podospora anserina, amyloid, filamentous fungi, maternal inheritance, heterokaryon incompatibility, cell death, epigenetics     

The genome sequence of Podospora anserina, a classic model fungus

The completed genome sequence of the coprophilous fungus Podospora anserina increases the sampling of fungal genomes. In line with its habitat of herbivore dung, this ascomycete has an exceptionally rich gene set devoted to the catabolism of complex carbohydrates.     

An attempt to establish a model for the rhythmic phenomena affecting the growth or sporulation of certain fungi

A simple two-variable mathematical model is proposed, able to acount for periodic variations relating to growth in Podospora anserina and fructification in Aspergillus niger. This basic model is open to generalization.     

Electrophoretic analysis of plasma membrane proteins of wild-type and differentiation-deficient mutant strains ofPodospora anserina

In the fungusPodospora anserina, themodC mutations inhibit the sexual female organ differentiation. Previous results have suggested that the plasma membrane of this mutant may be altered. Proteins solubilized from highly purified plasmalemma from a wild-type and threemodC mutant strains were studied by one- and two-dimensional electrophoresis. Isoelectric focusing revealed in the wild-type strain about 80 polypeptide spots whose molecular weight ranged from 15,000 to 80,000 daltons; 65% of the spots were between 20,000 and 60,000 daltons, and 60% were in the 6.5–7.5 pH zone. The only difference inprotein noted between wild-type and the threemodC mutants was that the threemodC mutants lacked a plasmalemma protein of M_r=42,000 and of pI=6.7. These results suggest a close relationship between plasma membrane proteins and differentiation in this eukaryotic organism.     

Ribosomal proteins in the fungus Podospora anserina: Evidence for an electrophoretically altered 60S protein in a cycloheximide resistant mutant

Molecular Genetics and Genomics - Proteins of cytoplasmic ribosomes of the Podospora anserina were analyzed by two dimentional gel electrophoresis. The numbers of proteins were estimated to be 28...     

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. A potential role for an 11 base-pair consensus sequence in the excision process

Three novel mitochondrial excision-amplification plasmids of Podospora anserina were identified and the excision-junction sites on the mitochondrial genome determined. All three plasmids were at least partially derived from a common region of the mitochondrial genome termed EcoRI-7 (E7). The entire 5651 base-pair sequence of E7 is presented. Included within this sequence are the E7-specific excision-junction sites of these novel plasmids, the localizations of nine tRNA genes, and the localization of a class I intron of the large rRNA mitochondrial gene. The E7 region contains the 3' portion of this large rRNA gene. Formation of these three novel plasmids as well as other previously described mitochondrial plasmids was found to be associated with the presence of an 11 base-pair consensus sequence, GGCGCAAGCTC, or its complementary sequence. A possible role for this consensus sequence and its complement in plasmid formation and the senescence process of Podospora is discussed. A possible role for the tRNA genes in plasmid formation is considered.     

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process

Summary We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.     

A Single Amino Acid Difference Is Sufficient to Elicit Vegetative Incompatibility in the Fungus Podospora Anserina

Vegetative incompatibility is known to limit heterokaryosis in filamentous fungi. It results from genetic differences between incompatible strains at specific loci. The proteins encoded by the two incompatible alleles het-s and het-S of the fungus Podospora anserina differ from each other by 14 amino acids. Two approaches have been used to identify how many and which of these differences are necessary to elicit incompatibility. Twelve alleles of the het-s locus of wild-type isolates of P. anserina and of the related species Podospora comata have been sequenced to determine the extent of the variability of genes controlling s and S specificities. Expression of hybrid het-s/het-S genes and site-specific mutagenesis revealed that the specificities of het-s and het-S are under the control of a limited number of amino acid differences. The results show that vegetative incompatibility between s and S strains can be attributed to a single amino acid difference in the proteins encoded by the het-s locus.     

The Podospora rmp1 gene implicated in nucleus-mitochondria cross-talk encodes an essential protein whose subcellular location is developmentally regulated

It has been previously reported that, at the time of death, the Podospora anserina AS1-4 mutant strains accumulate specific deleted forms of the mitochondrial genome and that their life spans depend on two natural alleles (variants) of the rmp1 gene: AS1-4 rmp1-2 strains exhibit life spans strikingly longer than those of AS1-4 rmp1-1. Here, we show that rmp1 is an essential gene. In silico analyses of eight rmp1 natural alleles present in Podospora isolates and of the putative homologs of this orphan gene in other filamentous fungi suggest that rmp1 evolves rapidly. The RMP1 protein is localized in the mitochondrial and/or the cytosolic compartment, depending on cell type and developmental stage. Strains producing RMP1 without its mitochondrial targeting peptide are viable but exhibit vegetative and sexual defects.     

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.