Marguerite Picard

Increased longevity of EF-1α high-fidelity mutants in Podospora anserina

Various translation initiation and elongation factors seem to participate in the control of the cellular proliferation and the ageing process in higher eukaryotes. Studies indicate that EF-1α, one of the translation elongation factors, may be one of the major components involved. We here present the cloning of the filamentous fungus P. anserina EF-1α encoding gene and show that strains bearing high fidelity mutations in the EF-1α gene have a drastically increased longevity as well as an impairment in sporulation. This suggests that EF-1α is involved in the sexual and senescence processes in lower eukaryotes, through the control of translational errors.

A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1‐1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1‐1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1‐1 also shows defects in ascospore germination and growth rate. GFP‐tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating‐type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

Abstract In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat− or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins.

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat−, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat− information. The transformants behave in crosses as do the reference mat+ or mat− strains, thus indicating that the transgenic mat+ and mat− are fully functional even when they have integrated at ectopic sites.

Lack of mitochondrial citrate synthase discloses a new meiotic checkpoint in a strict aerobe

Mitochondrial citrate synthase (mCS) is the initial enzyme of the tricarboxylic acid (TCA) cycle. Despite the key position of this protein in respiratory metabolism, very few studies have addressed the question of the effects of the absence of mCS in development. Here we report on the characterization of 15 point mutations and a complete deletion of the cit1 gene, which encodes mCS in the filamentous fungus Podospora anserina. This gene was identified genetically through a systematic search for suppressors of the metabolic defect of the peroxisomal pex2 mutants. The cit1 mutant strains exhibit no visible vegetative defects. However, they display an unexpected developmental phenotype: in homozygous crosses, cit1 mutations impair meiosis progression beyond the diffuse stage, a key stage of meiotic prophase. Enzyme assays, immunofluorescence and western blotting experiments show that the presence of the mCS protein is more important for completion of meiosis than its well‐known enzyme activity. Combined with observations made in budding yeast, our data suggest that there is a general metabolic checkpoint at the diffuse stage in eukaryotes.

The Podospora rmp1 gene implicated in nucleus-mitochondria cross-talk encodes an essential protein whose subcellular location is developmentally regulated

It has been previously reported that, at the time of death, the Podospora anserina AS1-4 mutant strains accumulate specific deleted forms of the mitochondrial genome and that their life spans depend on two natural alleles (variants) of the rmp1 gene: AS1-4 rmp1-2 strains exhibit life spans strikingly longer than those of AS1-4 rmp1-1. Here, we show that rmp1 is an essential gene. In silico analyses of eight rmp1 natural alleles present in Podospora isolates and of the putative homologs of this orphan gene in other filamentous fungi suggest that rmp1 evolves rapidly. The RMP1 protein is localized in the mitochondrial and/or the cytosolic compartment, depending on cell type and developmental stage. Strains producing RMP1 without its mitochondrial targeting peptide are viable but exhibit vegetative and sexual defects.

Overexpression of a human and a fungal ABC transporter similarly suppresses the differentiation defects of a fungal peroxisomal mutant but introduces pleiotropic cellular effects.

Among the peroxisome membrane proteins, some are required for peroxisome biogenesis (e.g. PEX2) while others are not, e.g. ABC (ATP‐binding cassette) transporters. Unexpectedly, overproduction of the peroxisomal ABC transporter PMP70 was found to be able to restore peroxisome biogenesis in mammalian pex2 mutant cell lines. In the filamentous fungus Podospora anserina, pex2 mutations not only impair peroxisome biogenesis but also cause a precise cell differentiation defect. Here, we show that both defects are partially suppressed by expression of the human cDNA encoding PMP70. In addition, PMP70 expression causes new developmental defects, different from those induced by pex2 mutations. We also show that overexpression of the P. anserina pABC1 gene, which encodes a peroxisomal ABC transporter, leads to similar effects. Taken together, our results demonstrate that: (i) the genetic relationship between PEX2 and PMP70, initially observed in mammals, has been conserved through evolution; (ii) the cell differentiation defect observed in the P. anserina pex2 mutants is indeed linked to impairment in peroxisome biogenesis; and (iii) unexpected detrimental cellular defects result from overproduction of peroxisomal ABC transporters.