Background: The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.

SummaryIn 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 .

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 ⌬PaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent proteinPaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in ⌬PaATG1 and ⌬PaATG8 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 ⌬PaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.Macroautophagy, generally referred to as autophagy, occurs coincidently with cell death in type II programmed cell death (PCD) (17). Autophagy has been first described as a cellular response to nutrient starvation. This intracellular catabolic system allows degradation and recycling of long-lived proteins and organelles in eukaryotic cells (44). During this process, the cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes (44). These vesicles then fuse by their outer membrane to the vacuolar/lysosomal membrane and deliver single-membrane vesicles called autophagic bodies inside the lumen of these organelles for degradation. The autophagic/vacuolar type of cell death (type II) is defined by morphological features that are the presence of autophagosomes or autophagic bodies inside the lysosome/vacuole degradative compartment with or without large vacuoles (8). Thus, type II PCD is defined as autophagic cell death, but this classification does not imply that autophagy directly contributes to cell death. In fact, two opposing views on the role of autophagy in type II PCD have emerged. Autophagy is either viewed as a medium of cellular demise or, on the contrary, as a prosurvival mechanism. Autophagy is also associated with various diseases in humans, including cancer and neurodegenerative disorders (25). Whether autophagy protects from or causes disease is also unclear (45).The molecular dissection of autophagy has been mostly performed in Saccharomyces cerevisiae and led to the identifica...