Jean Bernet

Protoplasmic incompatibility and female organ formation in Podospora anserina: Properties of mutations abolishing both processes

SummaryA genetic block, effective only with the addition of mutations in two genes, suppresses both the incompatibility process in strain confrontation (“barrage”) and the formation of female organs. Investigations on this block lead us to propose that it operates at the ribosomal level and prevents the translation of the messenger of a proteolytic enzyme, a postulated early protein in protoperithecia differenciation.

Polypeptide synthesis during protoplasmic incompatibility in the fungus Podospora anserina

In Podospora anserina, self-lysis resulting from the combination of the R and V incompatibility genes is accompanied by the appearance, in lysing cells, of specific enzyme activities, among which is a laccase exoenzyme, and by a quenching of ribonucleic acid synthesis. Present results show that the occurrence of the laccase is the result of de novo synthesis. By means of two-dimensional gel electrophoresis it was shown that the onset of self-lysis is accompanied by the immediate shut-off of more than 60% of the pre-existing normal polypeptide synthesis and the occurrence of at least 20 new polypeptides. The synthesis of these new polypeptides is active for several hours after the cessation of RNA synthesis, concurrently with the synthesis of about 30 normal polypeptides which is maintained. These modifications of protein synthesis are not accompanied by a concomitant variation in the level of polysomes. It is deduced that incompatibility genes are involved in the control of both transcription and translation.

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.

Intracellular and extracellular phenoloxidases in the fungus Podospora anserina: Effect of a constitutive mutation in a gene involved in their posttranscriptional control

SummaryThree phenoloxidases- and in particular phenoloxidase B, an enzyme found in cell extracts only in the cases of protoplasmic incompatibility and cell lysis where it occurs in association with a protease — were investigated in a wild type strain. Phenoloxidase B was present only in the culture filtrates, while the other two phenoloxidases were found in cell extracts and in the culture medium. Addition of casaminoacids and ammonium acetate to the culture medium produced opposite effects: under the first conditions, phenoloxidase B increased in being present only in the culture filtrates and in the second medium, like a second phenoloxidase (activity C), phenoloxidase B completely disappeared from the culture filtrates.A recessive mutation was isolated in a gene modB that resulted in the production of phenoloxidases B and C in higher amounts. The temperature dependence of the mutation modB allowed us to show that the formation of the phenoloxidases was 5 Fluorouracilresistant and cycloheximide sensitive, suggesting that constitutivity for these enzymes resulted from a derepression in their posttranscriptional control. Because of this derepression, phenoloxidase B not only increased in the culture medium (30x), but was found in cell extracts in association with a protease whose presence is suspected to produce a premature disintegration of the mycelium.The results are discussed from the point of view of the processes capable of regulating the production of exoenzymes and in relationship with the phenomenon of cell death normally affecting mycelia during ageing.

Incompatibility in the fungus Podospora anserina

SummaryIn the Ascomycete Podospora anserina the incompatibility reaction due to the interaction of non allelic genes exhibits some sensitivity to the antibiotic dihydrostreptomycin as well as to high levels of Magnesium. This incompatibility reaction can be suppressed or made more sensitive to the Magnesium or dihydrostreptomycin effect by mutations in the same genes mod1 and mod2. Properties of mutant strains suggest that mod1 and mod2 are ribosomal genes whose products seem to regulate, in a positive way for the first gene and in a negative way for the second gene, the translation of specific messengers, especially that of some proteolytic enzymes.

A Pleiotropic Mutation Affecting Protoperithecium Formation and Ascospore Outgrowth in Podospora anserina

A mutation designated modD, which was selected on the basis of the suppression of selflysis, produced a decrease in the density of protoperithecia and a deficiency in ascospore outgrowth at 26 °. Strains carrying the mutation were wild-type at 32°. Incubation of mutant ascospores revealed two abnormalities. First, the induction of ascospore outgrowth occurred only under a restricted range of conditions. Outgrowth of wild-type spores was normal down to 8 ° in a standard germination medium and could also be initiated in a non-germinating medium by the addition of actinomycin D or 5-fluorouracil. Mutant ascospores did not exhibit outgrowth in the presence of these two drugs or in a standard germination medium below 20 °. The second defect was the death of 80% of mutant ascospores when they were incubated in germination medium at 26 °. This killing was suppressed by β-phenylpyruvic acid, a protease inhibitor, and by a mutation (modC) which suppresses the lytic proteases associated with protoplasmic incompatibility and self-lysis. The killing of mutant spores was also suppressed by cycloheximide but was unaffected by actinomycin D and 5-fluorouracil.

Regulation of proteolytic enzymes inPodospora anserina: Selection and properties of self-lysing mutant strains

SummaryPrevious results showed that cell disintegration in the fungusPodospora anserina occured through the action of two proteases, enzymes whose messengers were normally latent during the extension stage of the thallus.We selected three mutant strains in which the constitutive activity of the protease messengers was expressed by an arrest of growth early in development (10 to 30 hours after spore germination) and a reaction of cell disintegration, in the thallus, suppressible with β-phenyl pyruvic acid, a protease inhibitor.The mutant character is recessive in one strain. In the case of the two strains in which the mutant trait is dominant, reversion studies have revealed that the deregulation resulted from the specific interaction between two genes and we have succeded in creating two non allelic incompatibility systems comparable to the non allelic gene interactions responsible for the incompatibility phenomena found between wild type races.We know, on the whole, that 11 loci are involved in the regulation of the proteases: five were revealed as incompatibility loci and six were discovered from investigations on four self-lysing mutant strains. It is suspected that all these genes act at the post-transcriptionnal level of the synthesis of specific proteolytic enzymes. We propose that the products of two genes act as “repressors” to prevent the protease messengers from being constitutively translated and that the products of the nine remaining genes exert a positive control by inducing translation, at the appropriate time, through the action of effectors resulting from specific interloci cooperation.

Protoplasmic Incompatibility and Cell Lysis in Podospora Anserina: Effect of β-phenyl Pyruvic Acid

Summary Previous results showed that the presence of nonallelic incompatible genes in a common cytoplasm induced, first, the formation of specific proteases thought to be responsible for cell destruction (B egueret and B ernet 1973) and second, a metabolic block resulting in a reduction of protein synthesis and the cessation of RNA production (L abarere et al. 1974). In vitro experiments show that β -phenyl pyruvic acid, a drug known to suppress cell disintegration without reversing the metabolic block, inhibits the proteases specific to lysing cells. Mutations have been selected that, in addition to the presence of β -phenyl pyruvic acid, resulted in the suppression of the metabolic block. We show that these mutations derive from a gene modA, whose first identified mutations suppressed the formation of the proteases and reversed the metabolic block. It is deduced that the metabolic block is not a secondary consequence of nonallelic gene interaction resulting, for instance, from the presence of the proteases specific to lysing cells. The results suggest instead that the inhibition of protein (and RNA) synthesis is, like the synthesis of the lytic proteases, the consequence of a process initiated by nonallelic gene interaction and involving as a co-factor the product of the gene modA.

A gene suppressing the allelic protoplasmic incompatibility specified by genes at five different loci in Podospora anserina

SUMMARY: Protoplasmic incompatibility (PI) in fungi is a phenomenon of immediate cell destruction resulting from the fusion of cells of unlike genotypes. In Podospora anserina, five loci contain genes determining PI as the result of allelic gene interactions. The present work shows that mutations of a gene called modD inhibit allelic PI irrespective of the locus responsible for the phenomenon. Other modD mutations show differential actions on the allelic interactions and on the expression of the two allelic incompatibility genes of the same locus. These results thus suggest that the modD gene product is involved in the trigger mechanism of allelic PI. The modD gene acts in differentiation: it has been previously shown to control proteolytic activities required for exit from stationary phase. There is thus a connection between this function and allelic PI. This leads to the suggestion that allelic incompatibility genes are involved in the control of stationary phase exit to promote differentiation.

Protoplasmic Incompatibility in Podospora anserina: Possible Involvement of the Plasma Membrane in the Trigger Mechanism

Two mutations in the modC locus of Podospora anserina, which abolish the process of cell lysis resulting from the combination of the R and V non-allelic incompatibility genes, have previously been shown to produce complete suppression of a laccase exoenzyme. In the present study it was found that, in addition, the modC mutants show a marked increase in the activity of an amino-acid oxidase (an exo- and endoenzyme), most of the increase (some 9- or 15-fold) being in the extracellular fraction. The modC mutant strains are hypersensitive to cycloheximide in vivo, but biochemical and genetical investigations suggested that the hypersensitivity is not due to effects on the ribosome. The modC(l) strain also shows increased resistance to chlorate, a specific sensitivity to acid (6.5) and neutral (7.5) pH, and significant hypersensitivity to methylammonium and thiourea. ModC cells accumulate a higher amount of the urea analogue [14C]thiourea. The pH sensitivity associated with the modC(l) mutation is specifically suppressed by the combination of the R and V incompatibility genes, and the RVmodC(l) strain is resistant to sorbose. The specific consequences of the modC(l) mutation and the suppression of some of its effects by the combination of the R and V incompatibility genes lead to the suggestion that protoplasmic incompatibility might be induced by action of the R/V initiating complex in association with the plasma membrane.