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PubMed2006

The peroxisomal import proteins PEX2, PEX5 and PEX7 are differently involved in Podospora anserina sexual cycle.

PEX5, PEX7 and PEX2 are involved in the peroxisomal matrix protein import machinery. PEX5 and PEX7 are the receptors for the proteins harbouring, respectively, a PTS1 and a PTS2 peroxisomal targeting sequence and cycle between the cytoplasm and the peroxisome. PEX2 belongs to the RING-finger complex located in the peroxisomal membrane and acts in protein import downstream of PEX5 and PEX7; it is therefore required for the import of both PTS1 and PTS2 proteins. We have shown previously that PEX2 deficiency leads to an impairment of meiotic commitment in the filamentous fungus Podospora anserina. Here we report that both PEX5 and PEX7 receptors are dispensable for this commitment but are needed for normal sexual cycle. Data suggest also a new role of PEX2 and/or the RING-finger complex in addition to their role in PTS1 and PTS2 import. Strikingly, Deltapex5 and Deltapex7 single and double knockout strains analyses indicate that Deltapex7 acts as a partial suppressor of Deltapex5 life cycle deficiencies. Moreover, contrary to pex2 mutants, Deltapex5 and Deltapex7 show mitochondrial morphological abnormalities.

Biological Transport↗
PubMed2005

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

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

Amino Acid Sequence↗
PubMed2005

DNA-binding specificity of the IDI-4 basic leucine zipper factor of Podospora anserina defined by systematic evolution of ligands by exponential enrichment (SELEX).

Heterokaryon incompatibility is a cell destruction process that occurs when fungal cells of unlike genotype fuse. In Podospora anserina, autophagy is engaged during cell death by incompatibility and a number of genes are induced at the transcriptional level. These genes are termed idi (induced during incompatibility) genes. Among these is idi-4, a gene encoding a basic leucine zipper (bZIP) factor. IDI-4 displays similarity to the GCN4/cross-pathway control (CPC) factors that control gene expression in response to amino acid starvation in fungi. The overexpression of idi-4 triggers autophagy, leads to cell death, and also increases the expression of a number of idi genes, in particular idi-7, a gene involved in autophagy. Herein, we determined the in vitro target sequence of IDI-4. We have purified the recombinant IDI-4 bZIP domain and show that this 83-amino-acid-long peptide dimerizes in vitro and adopts an alpha-helical fold. We have then used a systematic evolution of ligands by exponential enrichment procedure to identify the sequence bound by the IDI-4 bZIP domain. The IDI-4 binding site consensus sequence corresponds to the ATGANTCAT pseudopalindrome. IDI-4 binding sites are present in the promoter region of the idi-7 gene, and the bZIP IDI-4 peptide binds to the idi-7 promoter in vitro. The identified IDI-4 consensus binding sequence is very similar to the GCN4/CPC binding site, raising the possibility of an interplay and/or partial functional redundancy between IDI-4 and CPC-type bZIP factors in fungi.

Amino Acid Sequence↗
PubMed2006

OXPHOS Supercomplexes: respiration and life-span control in the aging model Podospora anserina.

Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. We have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life-span control and aging.

Aging↗
PubMed2004

Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina.

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.

Amino Acid Substitution↗
PubMed2004

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

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

Cell Nucleus↗
PubMed1998

Optional introns in mitochondrial DNA of Podospora anserina are the primary source of observed size polymorphisms.

The significant differences in mitochondrial genome size among seven races (B, E, M, T, U, W, and Y) of Podospora anserina have been found to be primarily due to the presence and/or absence of introns, including four introns not previously known to be optional. Information from physical mapping of races M and T, and sequence data from races A and s, was used to identify regions likely to contain insertions or deletions, which were then characterized using PCR and sequence analysis. Newly confirmed optional introns are the first intron of the large ribosomal RNA (LSUr1), the single intron of NADH dehydrogenase subunit 3 (ND3i1), the single intron in ATPase subunit 6 (ATPase6), and the fifth intron of cytochrome oxidase subunit I (COIi5). We have also found that race M exists in two forms as determined by mitochondrial DNA. These results bring to nine (including races A and s) the number of races characterized by mitochondrial intron content with a total of six known optional introns and one optional insertion. Eight of the nine races contain a distinct set of introns, providing a more reliable means for identification and comparison. The identification of optional mitochondrial introns in P. anserina may have evolutionary implications regarding the transfer and/or mobility of these introns.

Ascomycota↗
PubMed1998

Escape from Premature Death Due to Nuclear Mutations in Podospora anserina: Repeal versus Respite.

Premature death has been defined as a growth stoppage linked to the accumulation of specific deletions of the mitochondrial genome (mtDNA) in Podospora anserina. This occurs only in strains carrying the AS1-4 mutation which lies in a gene encoding a cytosolic ribosomal protein. Here we describe the isolation and genetic characterization of 10 nuclear mutations which either delay the appearance of this syndrome (respite from premature death) or cause a switch to the classical senescence process (repeal of premature death). These mutations lie in at least six genes. Some cause defects at the levels of ascospore germination, growth rates, and/or sensitivity toward inhibitors of protein syntheses. All modify the onset of senescence in wild-type (AS1+) strains. The role played by these genes is discussed with respect to the control of diseases due to mtDNA rearrangements in filamentous fungi. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Use of a linear plasmid containing telomeres as an efficient vector for direct cloning in the filamentous fungus Podospora anserina.

In Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi.

Ascomycota↗
PubMed1999

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.

Aging↗
PubMed1994

DNA double-strand break in vivo at the 3' extremity of exons located upstream of group II introns. Senescence and circular DNA introns in Podospora mitochondria.

In the filamentous fungus Podospora anserina, the unavoidable phenomenon of senescence is associated with the amplification of the first intron of the mitochondrial cox1 that accumulates as circular DNA molecules consisting of tandem repeats. This group II intron (cox1-i1 or alpha) is able to transpose and contains an open reading frame with significant amino acid similarity with reverse transcriptases. The generation of these intronic circular DNA molecules, their amplification and their involvement in the senescence process are unresolved questions. We demonstrate here that: (1) another group II intron, the fourth intron of gene cox1, cox1-i4, is also able to give precise DNA end to end junctions; (2) this intronic sequence can be found amplified during senescence, although to a lesser extent than cox1-i1; (3) the amplification of the DNA multimeric cox1-i1 molecules likely does not proceed by autonomous replication; (4) the generation of the DNA intronic circles does not require efficient intron splicing; (5) a DNA double-strand break occurs in vivo at the 3' extremity of the cox1-e1 and cox1-e4 exons preceding the group II introns that form circular DNAs. On the whole, these results show that the ability to form DNA circular molecules is a property of some group II introns and they demonstrate the occurrence of a specific DNA cleavage at or near the integration site of these group II introns. The results strongly suggest that this cleavage is involved in the formation of the group II intronic DNA circles and could also be involved in the phenomenon of group II intron homing.

Ascomycota↗
PubMed1991

Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes.

Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.

Ascomycota↗
PubMed1980

A new mutant form of the ribosomal protein L21 in the fungus Podospora anserina: identification of the structural gene for this protein.

A muation in the cyR1 gene of the fungus Podospora anserina confers resistance to cycloheximide and leads to an alteration of the 60S ribosomal protein L21 (Bégueret et al. 1977). Nine revertants of this mutant were isolated and the properties of these strains were analyzed. It was found that one revertant strain contains a new mutant form of L21. It is proposed that cyR1 gene is the structural gene for protein L21 and that the alteration of this protein is responsible for the resistance to cycloheximide in vivo.

Ascomycota↗
PubMed1979

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.

Ascomycota↗
PubMed1979

Dominant enhancer effect of the meiotic mei4 mutant on recombination frequencies restricted to linkage group VI in Podospora anserina.

A mutant which increases second division segregation (SDS) frequency of locus 110 (linkage group VI) was isolated. It was called mei4 because of its meiotic deficiency. The present paper deals with its effect on meiotic recombination when heterozygous. mei4 then only acts on linkage group VI. The SDS frequencies were increased for all markers used, except locus 5 located very close to the centromere. This quasi general enhancement results exclusively in an enlargement of map distance on linkage group VI's proximal part. Crosses involving three mutant genes allowed to check that the distances on the distal part were constant. This is due to a real lack of crossover frequency modification in this region and not to a change ofchiasma interference. Among the seven linkage groups of Podospora anserina, group VI exhibits several other particularities concerning meiotic recombination, especially a lower positive chiasma interference and a more regular crossover distribution, suggesting a particular recombination regulation.

Ascomycota↗
PubMed1981

Mutations affecting translational fidelity in the eucaryote Podospora anserina: characterization of two ribosomal restrictive mutations.

Fifty-nine mutations that restrict suppressor efficiency were selected in the fungus Podospora anserina using four different screening methods. Previous genetic analysis has shown that these antisuppressors lie in six loci and that they could be similar to ribosomal restrictive mutations known in Escherichia coli. The present study deals with the response of two of them, AS1-1 and AS6-1, to paromomycin and low temperature both in vivo and in vitro. The data demonstrate that ribosomes of the mutant and double-mutant strains are equally resistant to the ambiguity effect of paromomycin. These data are the first demonstration of mutations that increase translational fidelity in eucaryotic organism.

Ascomycota↗
PubMed1976

Genes inhibiting senescence in the ascomycete Podospora anserina.

Senescence occurs in all wild strains of Podospora anserina after continued growth. This syndrome can be inhibited by a synergistic interaction of two linked genes, incoloris and vivax. Whereas the wild strain starts to become senescent after 26 d and the mutants incoloris and vivax after 42 and 66 d respectively, the double mutant shows no signs of aging after culture for more than one year.

Ascomycota↗