Characterization of hch, the Podospora anserina homolog of the het-c heterokaryon incompatibility gene of Neurospora crassa

The het-c locus controls heterokaryon formation in Neurospora crassa. It is subject to balancing selection operating to maintain polymorphism at that locus in natural populations. We have isolated hch, the het-c homolog from the related species Podospora anserina (hch for het-c homolog), in order to determine if this gene also functions as a het gene in that species. The het-c and hch sequences are highly similar but differ in the region defining allele specificity in N. crassahet-c. Analysis of hch variability in 11 natural P. anserina isolates with different het genotypes revealed no polymorphism. This suggested that hch does not function as a het gene. However, heterologous expression of the N. crassa het-c ^PA allele in P. anserina triggers a growth defect reminiscent of the het-c incompatibility reaction. Received: 23 November 1999 / 7 March 2000     

Transformation to senescence with plasmid like DNA in the ascomycete Podospora anserina

Summary In the ascomycete Podospora anserina senescence through strain aging is under nucleo-cytoplasmic control and inducible in juvenile mycelia by an infective principle transferred after cytoplasmic contact via anastomoses. A specific DNA called plasmid-like (pl) DNA, present exclusively in aging mycelia, was found to be identical with this infective principle, since it was possible to transform juvenile protoplasts to senescence by using purified p1DNA. Therefore a specific function may be attributed to this ccc DNA. Its direct involvement in a genetically programed senescence is confirmed and its development as a vector for transfer of genetic information in eukaryotes can be undertaken.     

Genetics of translational fidelity in Podospora anserina: are all the genes involved in this ribosomal function identified?

Summary Su12-1 and su12-2 are two ribosomal suppressor mutations previously described in the fungus Podospora anserina. Revertants were isolated on the criteria of either improved growth at 27 °C (for su12-1) or suppression of the paromomycin hypersensitivity (for su12-2). Among 45 mutations lying outside the su12 locus, only one was found which defines a new antisuppressor locus, AS9. About 3/4 of these mutations are antisuppressor mutations localized in the previously identified AS6 and AS7 genes. While the AS6 mutations harbour diverse phenotypes, all the mutations lying in the AS7 gene lead to the same phenotypic alterations. In addition, two new su3 mutations were obtained and shown to display an antisuppressor effect on su12-1.     

Podospora anserina mutations inhibiting several developmental alternatives and growth renewal

Summary A mutation (modG) was selected on the basis of the suppression of a defect resulting from a mutation of modD gene. The modG mutation shows the same developmental consequences as modD mutations: the absence of protoperithecia and aerial hyphae, the non-renewal of growth from stationary cells and the non-germination of ascospores. Investigations of heterokaryotic mycelia showed that the action of modD and modG mutations on the formation of protoperithecia and aerial hyphae is autonomous. Furthermore, taking advantage of the thermosensitivity of a modD mutation, it was shown that the mutations have no incidence on cell differentiation prior to the achievement of stationary physiology. These findings lead to suggest the existence, in Podospora anserina, of a stage of cell totipotency, which we call the switch stage, that derives from the stationary state through the action of modD and mode genes. Accordingly, the switch stage would be the intermediate in the process of growth renewal from stationary cells and the basic stage for the selection of the developmental alternatives (aerial hyphae, protoperithecia ...) of Podospora mycelia.     

Evidence for giant linear plasmids in the ascomycete Podospora anserina

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1.     

Electrophoretic karyotype of the ascomycete Podospora anserina

Summary Fractionation of intact chromosomes of the ascomycete Podospora anserina by contour-clamped homogenous electric field (CHEF) gel electrophoresis resulted in the resolution of five distinct chromosomal bands. Two of these bands migrated as doublets. Using chromosomal standards from Schizosaccharomyces pombe we estimated the size of the P. anserina genome to about 33.4 megabases (Mb). By heterologous hybridization of fractionated chromosomes the rDNA locus was identified on one of the two chromosomes migrating at about 4.9 Mb.     

Transformation of Podospora anserina with a dominant resistance gene

Summary The Podospora anserina immortal mutant incoloris, vivax was transformed to benomyl resistance with a -tubuline gene from a resistant Neurospora crassa strain. The transforming plasmid was integrated into the genome of the transformants, and subsequent Southern analysis and retransformation experiments provided no evidence for autonomous replication. Non-homologous integration was demonstrated in some of the transformants. Resistance to benomyl varied widely among the transformants and was conserved after the transformants were grown on non-selective medium.     

Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.     

Gene deletion and allelic replacement in the filamentous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis>

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

Reversion of a long-living,undifferentiated mutant of Podospora anserina by copper

The Podospora anserina nuclear mutant grisea displays an undifferentiated growth phenotype (diminished production of aerial hyphae), is female sterile (lack of perithecia), has a prolonged life-span compared to the wild-type strain, and lacks detectable phenoloxidase (laccase and tyrosinase) activity. Reversion of all of these characteristics to those of the wild-type phenotype was accomplished by supplementing the growth medium with extra amounts of copper salts. These results indicate that the primary defect of the grisea strain is in its copper uptake and/or distribution in the cells.     

The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene

Summary In the ascomycete Podospora anserina strain ageing (senescence) is caused by a mitochondrial plasmid. In juvenile mycelia it is an integral part of the mtDNA and becomes liberated during ageing. The nucleotide sequence of this plasmid and of its flanking regions was determined. It consists of 2,539 by and contains an un identified reading frame (URF) originating in the adjacent mtDNA upstream of excision point 1. Within the URF a putative 48 by autonomously replicating sequence (ars) was identified. At both excision sites of the plasmid there are two short nonidentical interrupted palindromes and a few base pairs apart from these palindromes, both upstream and downstream, two short inverted repeats are localised. The experimental data make it evident that the mt plasmid is an intron of the cytochrome c oxidase gene (subunit I) which may be excised at the DNA level and thus become the mobile infective agent causing senescence. The concept of this mobile intron and current hypotheses concerning the relationship between introns and transposons are stressed.     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.     

Electrophoretical and immunological comparison of the ribosomal proteins of the fungus Podospora anserina and the yeast Saccharomyces cerevisiae

Summary The ribosomal proteins of two ascomycetes Podospora anserina and Saccharomyces cerevisiae, were compared by two dimensional electrophoresis in two different gel systems and we found only five pairs of proteins which have kept homologous physico-chemical properties under these conditions. An immunological analysis was performed by radioimmunodetection of proteins blotted on nitrocellulose sheet after separation by electrophoresis, with four sera directed against the r-proteins of each subunit of these fungi. So, we pointed out many common antigenic sites present on proteins which do not co-migrate except for yeast L3 and L1 of P. anserina which have the same properties.     

Despite mtDNA polymorphism the mobile intron (plDNA) of the COI gene is present in ten different races of Podospora anserina

Summary Polymorphism of mitochondrial DNA (mtDNA) from ten different geographical races of Podospora anserina was characterized by means of restriction and hybridization analysis. Our data reveals that the mobile intron (plDNA), first characterized by DNA sequencing in strain s (Osiewacz and Esser 1984), is present in all of the races investigated. Therefore, we conclude that the mobile intron is not an optional intron of the COI gene in wild type strains. In addition, the detailed physical and genetical mapping of mtDNA from three different races with different lifespans leads us to propose that length polymorphism can result due to the lack or presence of intron sequences of structural mitochondrial genes. In view of our results we discuss the function of the mobile intron during senescence in P. anserina.     

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency

Summary The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5 non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5 regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.     

Isolation of the two allelic incompatibility genes s and S of the fungus Podospora anserina

Summary The two allelic genes s and S are responsible for heterogenic incompatibility between wild type strains of the fungus Podospora anserina. The s gene has been cloned by SIB selection and expression in a strain containing a neutral allele of this locus. The S gene was isolated from a genomic library using the DNA of the s locus as a probe. The physical map of the DNA fragments carrying the two genes are highly dissimilar and restriction polymorphism exists at the s locus between s and S strains. Nevertheless, homology between the two alleles was revealed by cross-hybridization at the DNA and RNA levels.     

Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants

Summary To develop a transformation system in the filamentous fungus Podospora anserina we have selected ura5 mutants deficient in orotidylic acid pyrophosphorylase using a positive screening. These mutants could be transformed to prototrophy by an hybrid vector carrying the ura5 gene of this organism. The properties of the transformants have been analysed. In most cases integration of the transforming vector occurred outside the ura5 locus and frequently repeated tandem copies of the vector were found. Reversion of the transformants could also be selected and we found that it can occur by exact or only partial excision of the integrated vector.Abbreviations bp base pairs - kb 1,000 bp - EtBr ethidium bromide - PEG polyethyleneglycol