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.     

The incompatibility relationships between geographical races of Podospora anserina

Summary The physiological action of heterogenic incompatibility between the geographical races s and S, which differ by a single gene and a corresponding cytoplasmic factor, has been studied. Microscopical, cytochemical, and enzymatic investigations have revealed that the cell disintegration occurring after anastomosis of incompatible hyphae under the immediate influence of the cytoplasmic factors leads to a destruction of cellular compartments which is followed by a liberation of catabolic enzymes. These incidents are counteracted by regenerative processes.Supported with grants from the Deutsche Forschungsgemeinschaft, Bad Godesberg, and from the Landesamt für Forschung, Düsseldorf.     

The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.     

The [Het-s] prion of Podospora anserina and its role in heterokaryon incompatibility

[Het-s] is a prion from the filamentous fungus Podospora anserina and corresponds to a self-perpetuating amyloid aggregate of the HET-s protein. This prion protein is involved in a fungal self/non-self discrimination process termed heterokaryon incompatibility corresponding to a cell death reaction occurring upon fusion of genetically unlike strains. Two antagonistic allelic variants of this protein exist: HET-s, the prion form of which corresponds to [Het-s] and HET-S, incapable of prion formation. Fusion of a [Het-s] and HET-S strain triggers the incompatibility reaction, so that interaction of HET-S with the [Het-s] prion leads to cell death. HET-s and HET-S are highly homologous two domain proteins with a N-terminal globular domain termed HeLo and a C-terminal unstructured prion forming domain (PFD). The structure of the prion form of the HET-s PFD has been solved by solid state NMR and corresponds to a very well ordered β-solenoid fold with a triangular hydrophobic core. The ability to form this β-solenoid fold is retained in a distant homolog of HET-s from another fungal species. A model for the mechanism of [Het-s]/HET-S incompatibility has been proposed. It is believe that when interacting with the [Het-s] prion seed, the HET-S C-terminal region adopts the β-solenoid fold. This would act as a conformational switch to induce refolding and activation of the HeLo domain which then would exert its toxicity by a yet unknown mechanism.     

The phenoloxidases of the Ascomycete Podospora anserina

Summary In order to learn the internal conditions for the production of the various phenoloxidases produced by the Ascomycete Podospora anserina the wild strain has been grown under controlled conditions in a fermenter for a period of 34 days. Samples were withdrawn at regular intervals and assayed for mycelial yield and intra- and extracellular phenoloxidase production.Maximal yield was obtained at the following age of the culture: Mycelial production 9 d, tyrosinase 4 d, the high molecular weight laccase I between 9 and 19 d. The low molecular weight laccases II and III, initially present in medium concentrations, dropped to an early minimum after 4 days, followed by an increase with a maximum in the late autolytic phase.The changes in the phenoloxidase spectrum and the antiparallel production curve for the high molecular weight against the low molecular weight laccases are discussed in relation to the earlier observed genetical and physiological control of phenoloxidase synthesis and in relation to the possibility of laccase I being composed of active subunits of low molecular weight laccases.With support of the Deutsche Forschungsgemeinschaft, Bad Godesberg (Germany).     

The phenol oxidases of the ascomycete Podospora anserina

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (K _M) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns.Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak.Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o-and p-diphenols, low affinity for m-phenols. The maximal velocity remains largely uninfluenced.This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.     

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.     

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.     

Ribosomal suppressors and antisuppressors in Podospora anserina: Altered susceptibility to paromomycin and relationships between genetic and phenotypic suppression

Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and their properties suggest that they could be ribosomal mutants involved in the control of translational fidelity. In this paper we present results concerning relationships between these mutants and paromomycin, an aminoglycoside antibiotic known to stimulate translational errors. The mutants were found to manifest an altered growth sensitivity to this drug as compared with the wild-type strain: Most of the suppressors were more sensitive and, in contrast, most of the antisuppressors were more resistant to paromomycin. Moreover, phenotypic suppression of an auxotrophic mutation by paromomycin was observed only if a suppressor and an antisuppressor had been introduced in the strain. These results suggest that ambiguity levels could be altered in the suppressor and antisuppressor strains. In addition, paromomycin was shown to abolish sporulation, which suggests relationships between mistranslation and a step of cellular differentiation.This work was supported by a DGRST grant and by a NATO grant.     

The genome sequence of Podospora anserina, a classic model fungus

The completed genome sequence of the coprophilous fungus Podospora anserina increases the sampling of fungal genomes. In line with its habitat of herbivore dung, this ascomycete has an exceptionally rich gene set devoted to the catabolism of complex carbohydrates.     

Chromosomal and extrachromosomal control of senescence in the ascomycete Podospora anserina.

Summary In Podospora anserina senescence leading to cellular death occurs regularly after prolonged vegetative propagation. However, the life span of this ascomycete may be extended by various means:I. Mutations in at least 8 morphogenetic genes belonging to 4 linkage groups postpone drastically or even prevent in certain pairwise combinations (e.g. i viv) the onset of senescence. 2. Inhibitors of mt DNA and of mitochondrial protein synthesis show a life prolonging effect when added in low concentrations to the growth medium. 3. A similar effect was found when mycelia were fed exclusively on non repressive carbon sources.Whereas the anti-aging effect of specific mutated genes is rather permanent, the life prolonging action of the inhibitors and carbon sources is restricted and temporary. These substances have no long lasting effect, since after their removal from the medium aging proceeds.Physiological experiments have further shown the existence of three phases in the life span of Podospora anserina. During the juvenile phase aging is prevented by all of these compounds; during the presenescent phase aging is prevented by inhibitors of mt DNA only, and during the senescent phase aging is irreversible.Senescence may be induced in juvenile protoplasts by DNA extracted from senescent mycelia. This, together with the well known fact that senescence is extrachromosomically inherited, points to extrachromosomal DNA as the causative agent of senescence. This kind of DNA may be connected with or perhaps located in the mitochondria.Collectively, the data are consistent in showing that the syndrome of senescence in Podospora anserina is controlled by a chromosomal-extrachromosomal is controlled by a chromosomal-extrachromosomal interaction. In this system, extrachromosomal DNA, perhaps a mt DNA, is identical with the infectious principle initiating the decay of the cell, and nuclear genes supervise its expression.     

HET-E and HET-D belong to a new subfamily of WD40 proteins involved in vegetative incompatibility specificity in the fungus Podospora anserina

Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2(Y) gene was isolated and shown to have strong similarity with the previously described het-e1(A) gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted beta-propeller structure defined by this domain may confer the incompatible interaction specificity.     

Cloning and characterization of the rDNA repeat unit of Podospora anserina

Summary DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.