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.     

Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina

In filamentous fungi, a programmed cell death (PCD) reaction occurs when cells of unlike genotype fuse. This reaction is caused by genetic differences at specific loci termed het loci (for heterokaryon incompatibility). Although several het genes have been characterized, the mechanism of this cell death reaction and its relation to PCD in higher eukaryotes remains largely unknown. In Podospora anserina, genes induced during the cell death reaction triggered by the het-R het-V interaction have been identified and termed idi genes. Herein, we describe the functional characterization of one idi gene (idi-1) and explore the connection between incompatibility and the response to nutrient starvation. We show that IDI-1 is a cell wall protein which localizes at the septum during normal growth. We found that induction of idi-1 and of the other known idi genes is not specific of the incompatibility reaction. The idi genes are induced upon nitrogen and carbon starvation and by rapamycin, a specific inhibitor of the TOR kinase pathway. The cytological hallmarks of het-R het-V incompatibility (increased septation, vacuolization, coalescence of lipid droplets, induction of autophagy, and cell death) are also observed during rapamycin treatment. Globally the cytological alterations and modifications in gene expression occurring during the incompatibility reaction are similar to those observed during starvation or rapamycin treatment.     

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.     

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.     

The fungus-specific HET domain mediates programmed cell death in Podospora anserina

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (het genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic het-C/het-E incompatibility system is the best-characterized incompatibility system in Podospora anserina. Cell death is triggered by interaction of specific alleles of het-C, encoding a glycolipid transfer protein, and het-E, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from het-E results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of het-C. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.     

Fungal incompatibility: Evolutionary origin in pathogen defense?

In fungi, cell fusion between genetically unlike individuals triggers a cell death reaction known as the incompatibility reaction. In Podospora anserina, the genes controlling this process belong to a gene family encoding STAND proteins with an N‐terminal cell death effector domain, a central NACHT domain and a C‐terminal WD‐repeat domain. These incompatibility genes are extremely polymorphic, subject to positive Darwinian selection and display a remarkable genetic plasticity allowing for constant diversification of the WD‐repeat domain responsible for recognition of non‐self. Remarkably, the architecture of these proteins is related to pathogen‐recognition receptors ensuring innate immunity in plants and animals. Here, we hypothesize that these P. anserina incompatibility genes could be components of a yet‐unidentified innate immune system of fungi. As already proposed in the case of plant hybrid necrosis or graft rejection in mammals, incompatibility could be a by‐product of pathogen‐driven divergence in host defense genes.     

Two allelic genes responsible for vegetative incompatibility in the fungus Podospora anserina are not essential for cell viability

Summary Vegetative incompatibility is a lethal reaction that destroys the heterokaryotic cells formed by the fusion of hyphae of non-isogenic strains in many fungi. That incompatibility is genetically determined is well known but the function of the genes triggering this rapid cell death is not. The two allelic incompatibility genes, s and S, of the fungus Podospora anserina were characterized. Both encode 30 kDa polypeptides, which differ by 14 amino acids between the two genes. These two proteins are responsible for the incompatibility reaction that results when cells containing s and S genes fuse. Inactivation of the s or S gene by disruption suppresses incompatibility but does not affect the growth or the sexual cycle of the mutant strains. This suggests that these incompatibility genes have no essential function in the life cycle of the fungus.