Corinne Clavé

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.

Accelerated Cell Death in Podospora Autophagy Mutants

ABSTRACT 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 ΔPaATG1 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 ΔPaATG1 and ΔPaATG8 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 ΔPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.

Vegetative incompatibility in filamentous fungi: het genes begin to talk

Somatic or vegetative incompatibility is widespread in filamentous fungi. It prevents the coexistence of genetically different nuclei within a common cytoplasm. Cloning the het genes that control this process has been achieved in several species. This has provided essential information on the function of the genes in the biology of fungi and has also led to the formulation of models that may explain similar phenomena in other organisms.

Rapamycin Mimics the Incompatibility Reaction in the Fungus Podospora anserina

ABSTRACT 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.

Characterization of IDI-4, a bZIP transcription factor inducing autophagy and cell death in the fungus Podospora anserina

In filamentous fungi a cell death reaction occurs when hyphae of unlike genotype fuse. This phenomenon is referred to as heterokaryon incompatibility. In Podospora anserina, this cell death reaction was found to be associated with the transcriptional induction of a set of genes termed idi genes (for induced during incompatibility) and activation of autophagy. Herein, we describe the characterization of idi‐4, a novel idi gene encoding a bZIP transcription factor. Expression of idi‐4 is induced during cell death by incompatibility and in various stress conditions. Inactivation of idi‐4 by gene replacement does not suppress incompatibility but we show that overexpression of idi‐4 triggers cell death. Strains which undergo idi‐4‐induced cell death display cytological hallmarks of cell death by incompatibility notably induction of autophagy. We also report that increased expression of idi‐4 leads to transcriptional induction of other idi genes such as idi‐7, the orthologue of the yeast ATG8 autophagy gene. Together these results establish IDI‐4 as one of the transcription factor regulating autophagy and cell fate in Podospora.

Podospora anserina target of rapamycin

We have isolated the Podospora anserinaTOR gene. The PaTOR protein displayed strong identities with TOR proteins from other eukaryotes especially in the FRB domain and the kinase domain. Genome analysis suggests that a single TOR gene exists in Podospora. The serine residue known to be one site of missense mutations conferring rapamycin resistance in other organisms is conserved in the PaTOR protein (S1895). A PaTOR-S1895R mutated allele has been constructed and introduced in the wild-type strain, as expected strains expressing the PaTOR-S1895R gene become resistant to rapamycin. The dominance of the PaTOR-S1895R allele indicates that apparently the mutation does not impair the kinase activity. We confirm that all cytological modifications associated with rapamycin treatment in Podospora are indeed mediated by PaTOR. We conclude that the PaTOR gene is likely to be essential and that rapamycin treatment might be useful to further investigate rapamycin-sensitive TOR functions in Podospora and especially newly identified rapamycin-sensitive functions such as the autophagy-independent control of vacuole remodeling and septation.

Two structurally similar fungal prions efficiently cross‐seed in vivo but form distinct polymers when coexpressed

HET‐s is a prion protein of the filamentous fungus Podospora anserina. An orthologue of this protein, called FgHET‐s has been identified in Fusarium graminearum. The region of the FgHET‐s protein corresponding to the prion forming domain of HET‐s, forms amyloid fibrils in vitro. These fibrils seed HET‐s(218–289) fibril formation in vitro and vice versa. The amyloid fold of HET‐s(218–289) and FgHET‐s(218–289) are remarkably similar although they share only 38% identity. The present work corresponds to the functional characterization of the FgHET‐s(218–289) region as a prion forming domain in vivo. We show that FgHET‐s(218–289) is capable of prion propagation in P. anserina and is able to substitute for the HET‐s PFD in the full‐length HET‐s protein. In accordance with the in vitro cross‐seeding experiments, we detect no species barrier between P. anserina and F. graminearum PFDs. We use the yeast Saccharomyces cerevisiae as a host to compare the prion performances of the two orthologous PFDs. We find that FgHET‐s(218–289) leads to higher spontaneous prion formation rates and mitotic prion stability than HET‐s(218–289). Then we analysed the outcome of HET‐s(218–289)/FgHET‐s(218–289) coexpression. In spite of the cross‐seeding ability of HET‐s(218–289) and FgHET‐s(218–289), in vivo, homotypic polymerization is favoured over mixed fibril formation.

NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi

Significance NOD-like receptors (NLRs) are fundamental components of plant and animal innate immune systems. Some fungal proteins with NLR-like architecture are involved in an allorecognition process that results in cell death, termed heterokaryon incompatibility. A role for fungal NLR-like proteins in pathogen defense has also been proposed. Here, we show that a fungal NLR-like protein, patatin-like phospholipase-1 (PLP-1), monitors the essential SNARE protein SEC-9 in two distantly related fungal species, Neurospora crassa and Podospora anserina. Both plp-1 and sec-9 are highly polymorphic in fungal populations and show evidence of balancing selection. This study provides biochemical evidence that fungal NLRs function similar to NLRs in plants and animals, indicating that these fundamental players of innate immunity evolved independently in all three kingdoms. In plants and metazoans, intracellular receptors that belong to the NOD-like receptor (NLR) family are major contributors to innate immunity. Filamentous fungal genomes contain large repertoires of genes encoding for proteins with similar architecture to plant and animal NLRs with mostly unknown function. Here, we identify and molecularly characterize patatin-like phospholipase-1 (PLP-1), an NLR-like protein containing an N-terminal patatin-like phospholipase domain, a nucleotide-binding domain (NBD), and a C-terminal tetratricopeptide repeat (TPR) domain. PLP-1 guards the essential SNARE protein SEC-9; genetic differences at plp-1 and sec-9 function to trigger allorecognition and cell death in two distantly related fungal species, Neurospora crassa and Podospora anserina. Analyses of Neurospora population samples revealed that plp-1 and sec-9 alleles are highly polymorphic, segregate into discrete haplotypes, and show transspecies polymorphism. Upon fusion between cells bearing incompatible sec-9 and plp-1 alleles, allorecognition and cell death are induced, which are dependent upon physical interaction between SEC-9 and PLP-1. The central NBD and patatin-like phospholipase activity of PLP-1 are essential for allorecognition and cell death, while the TPR domain and the polymorphic SNARE domain of SEC-9 function in conferring allelic specificity. Our data indicate that fungal NLR-like proteins function similar to NLR immune receptors in plants and animals, showing that NLRs are major contributors to innate immunity in plants and animals and for allorecognition in fungi.