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.     

Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina

NADPH oxidases are enzymes that produce reactive oxygen species (ROS) using electrons derived from intracellular NADPH. In plants and mammals, ROS have been proposed to be second messengers that signal defence responses or cell proliferation. By inactivating PaNox1 and PaNox2, two genes encoding NADPH oxidases, we demonstrate the crucial role of these enzymes in the control of two key steps of the filamentous fungus Podospora anserina life cycle. PaNox1 mutants are impaired in the differentiation of fruiting bodies from their progenitor cells, and the deletion of the PaNox2 gene specifically blocks ascospore germination. Furthermore, we show that PaNox1 likely acts upstream of PaASK1, a MAPKKK previously implicated in stationary phase differentiation and cell degeneration. Using nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) assays, we detect a regulated secretion of both superoxide and peroxide during P. anserina vegetative growth. In addition, two oxidative bursts are shown to occur during fruiting body development and ascospore germination. Analysis of mutants establishes that PaNox1, PaNox2, and PaASK1, as well as a still unknown additional source of ROS, modulate these secretions. Altogether, our data point toward a role for NADPH oxidases in signalling fungal developmental transitions with respect to nutrient availability. These enzymes are conserved in other multicellular eukaryotes, suggesting that early eukaryotes were endowed with a redox network used for signalling purposes.     

Peroxisomal ABC transporters and beta-oxidation during the life cycle of the filamentous fungus Podospora anserina

ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

repa, a repetitive and dispersed DNA sequence of the filamentous fungus Podospora anserina.

The sequences of homologous DNA regions of two wild-type strains of the fungus Podospora anserina, revealed in one strain the presence of a 349bp insertion leading to a RFLP. This DNA sequence is repeated in the genome and some of its locations are different in various wild-type strains. This DNA element exhibits structural similarities with the yeast solo delta, sigma or tau elements.     

Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts

From a genomic library of the filamentous fungus Podospora anserina, we have cloned a 4.9-kb fragment which complements an Escherichia coli mutant strain deficient for orotidylic acid pyrophosphorylase (pyrE gene). The recombinant plasmid pPAura5 also transforms to prototrophy a mutant strain of P. anserina carrying a mutation in the ura5 gene and lacking OMPppase activity.     

The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains

We have sequenced the ura5 gene of the filamentous fungus Podospora anserina. The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme. This comparison shows extensive blocks of homology. The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene. We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt). By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain.     

MOD-D, a Galpha subunit of the fungus Podospora anserina, is involved in both regulation of development and vegetative incompatibility.

Cell death via vegetative incompatibility is widespread in fungi but molecular mechanism and biological function of the process are poorly understood. One way to investigate this phenomenon was to study genes named mod that modified incompatibility reaction. In this study, we cloned the mod-D gene that encodes a Galpha protein. The mod-D mutant strains present developmental defects. Previously, we showed that the mod-E gene encodes an HSP90. The mod-E1 mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. Moreover, we isolated the PaAC gene, which encodes an adenylate cyclase, as a partial suppressor of the mod-D1 mutation. Our previous results showed that the molecular mechanisms involved in vegetative incompatibility and developmental pathways are connected, suggesting that vegetative incompatibility may result from disorders in some developmental steps. Our new result corroborates the involvement of mod genes in signal transduction pathways. As expected, we showed that an increase in the cAMP level is able to suppress the defects in vegetative growth due to the mod-D1 mutation. However, cAMP increase has no influence on the suppressor effect of the mod-D1 mutation on vegetative incompatibility, suggesting that this suppressor effect is independent of the cAMP pathway.     

Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region.     

A homolog of the proteasome-related RING10 gene is essential for yeast cell growth.

Proteasomes are intracellular protein complexes displaying multiproteolytic activities. These complexes have been implicated in the antigen degradation process that generates peptides associated with the major histocompatibility complex (MHC) class-I molecule. RING10 and RING12 are genes encoded by the class-II region of the human MHC that have sequence homology to proteasome-encoding genes. We have identified a yeast gene, called PRG1, that encodes a protein predicted to contain 55.6% sequence identity to 80% of the RING10 gene product. Genomic disruption of PRG1 revealed that it is essential for yeast cell growth. These data strongly indicate that the antigen-processing system present in vertebrates evolved from a basic cellular process present in all organisms.     

The fission yeast homologue of Glel is essential for growth and involved in mRNA export

We have isolated Glel homologue (named as spglel) as a partial multicopy suppressor of the synthetic lethality of rael-167 elfl-21 in fission yeast Schizosaccharomyces pombe. The spglel is also able to complement partially temperature-sensitive phenotype of rael-167 only at a lower restrictive temperature. The spglel gene contains one intron and encodes a 480 amino-acid protein with predicted molecular weight of 56.2 kDa. We showed that spglel gene is essential for vegetative growth and functional Glel-GFP protein is localized mainly in NPC. The accumulation of poly(A)(+) RNA in the nucleus is exhibited when expression of spglel is repressed or over-expressed. These results suggest that the spGle1 protein is also involved in mRNA export in fission yeast.     

Informational suppressor alleles of the eEF1A gene, fertility and cell degeneration in Podospora anserina

Mutations that increase readthrough at a UGA stop codon (informational suppressor mutations) were created in the gene (AS4) that encodes translation elongation factor eEF1A in the filamentous fungus Podospora amserina. The results strongly suggest that the net charge of the eEF1A protein controls the accuracy of translation. Physiological analysis of the mutant strains shows that some of the alleles dominantly increase life span, while only one drastically modifies fertility. This exceptional allele (AS4-56) causes a wide array of phenotypes, including a new growth cessation phenomenon that is different from Senescence or Crippled Growth, previously known degenerative syndromes that are both controlled by AS4. The data emphasise the fact that eEF1A exerts a complex control over cellular physiology.     

A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina

We present here the properties of a complex III loss-of-function mutant of the filamentous fungus Podospora anserina. The mutation corresponds to a single substitution in the second intron of the gene cyc1 encoding cytochrome c(1), leading to a splicing defect. The cyc1-1 mutant is long-lived, exhibits a defect in ascospore pigmentation, has a reduced growth rate and a reduced ROS production associated with a stabilisation of its mitochondrial DNA. We also show that increased longevity is linked with morphologically modified mitochondria and an increased number of mitochondrial genomes. Overexpression of the alternative oxidase rescues all these phenotypes and restores aging. Interestingly, the absence of complex III in this mutant is not paralleled with a deficiency in complex I activity as reported in mammals although the respiratory chain of P. anserina has recently been demonstrated to be organized according to the "respirasome" model.