The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids.     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids. Received: 13 November 1997 / Accepted: 17 February 1998     

Mitochondrial DNA rearrangements are correlated with a delayed amplification of the mobile intron (plDNA) in a long-lived mutant of Podospora anserina

A new long-lived mutant of Podospora anserina has been isolated and characterized. Its longevity is maternally inherited as revealed by reciprocal crosses. A molecular analysis resulted in the identification of an amplified DNA species (designated pAL2-1) with homology to mitochondrial DNA (mtDNA). The presence of this DNA species is correlated with mtDNA rearrangements and a delayed amplification of the mobile intron (plDNA).     

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.     

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.     

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassahet-c vegetative incompatibility locus, and its use as a population marker

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

Sir2-independent life span extension by calorie restriction in yeast

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.     

Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.     

A mitochondrial DNA rearrangement and three new mitochondrial plasmids from long-lived strains of Podospora anserina

The excision-junction sites of a mtDNA rearrangement of a long-lived strain of Podospora anserina, Mn19, were cloned and sequenced. Analysis of sequence and hybridization data lead to the conclusion that the Mn19 mtDNA consists of two nonoverlapping circular molecules. Three plasmids, LMt-2, LMt-3, and LMt-4, cloned from long-lived progeny of crosses between the Mn19 strain and wild type were cloned and sequenced. These plasmids share features and excision-junction sites with previously described longevity and senescence plasmids. The Mn19 mtDNA rearrangement and plasmids LMt-2, LMt-3, and LMt-4 are described. The possible significance of similarities to previously described plasmids is discussed.     

Sir2 blocks extreme life-span extension

Sir2 is a conserved deacetylase that modulates life span in yeast, worms, and flies and stress response in mammals. In yeast, Sir2 is required for maintaining replicative life span, and increasing Sir2 dosage can delay replicative aging. We address the role of Sir2 in regulating chronological life span in yeast. Lack of Sir2 along with calorie restriction and/or mutations in the yeast AKT homolog, Sch9, or Ras pathways causes a dramatic chronological life-span extension. Inactivation of Sir2 causes uptake and catabolism of ethanol and upregulation of many stress-resistance and sporulation genes. These changes while sufficient to extend chronological life span in wild-type yeast require severe calorie restriction or additional mutations to extend life span of sir2Delta mutants. Our results demonstrate that effects of SIR2 on chronological life span are opposite to replicatve life span and suggest that the relevant activities of Sir2-like deacetylases may also be complex in higher eukaryotes.     

Genetic and Molecular Analysis of a Long-Lived Strain of Podospora Anserina

A genetic and molecular analysis of a long-lived strain of Podospora anserina, Mn19, was undertaken to detect mutations in genes responsible for senescence. In crosses between Mn19 and wild type about 15% of the progeny were long-lived, regardless of the female parent. Molecular analysis of the long-lived progeny showed that none of the strains inherited a mtDNA rearrangement characteristic of the Mn19 parent. Instead, all long-lived strains initially inherited wild-type mtDNA. Over time the mtDNA of most long-lived strains underwent rearrangements, deletions and amplifications. The change over time in the presence of two previously characterized plasmids associated with either senescence or longevity was monitored. Crosses between Mn19 and its long-lived progeny also yielded only a small percent of individuals recovering from senescence. Analysis of mtDNA from crosses suggests that wild-type mtDNA from the paternal parent can be selected over mtDNA from the maternal parent. The life span phenotypes of progeny were not consistent with the hypothesis that mutations in a few nuclear genes were responsible for longevity.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina

Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.     

Multipartite structure of mitochondrial DNA in a fungal longlife mutant

Rearrangements in the mitochondrial genome of the longlife mutant ex1 of the ascomycete Podospora anserina have led to a heterogeneous population of subgenomic molecules. The restriction maps of individual subcircles were established using overlapping recombinant lambda phages isolated from an ex 1 mtDNA/EMBL3 library. The formation of the subcircles and the resulting multipartite organization of the ex1 mtDNA are discussed.     

Spore-killing meiotic drive factors in a natural population of the fungus Podospora anserina

In fungi, meiotic drive is observed as spore killing. In the secondarily homothallic ascomycete Podospora anserina it is characterized by the abortion of two of the four spores in the ascus. We have identified seven different types of meiotic drive elements (Spore killers). Among 99 isolates from nature, six of these meiotic drive elements occurred in a local population. Spore killers comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands, sampled from 1991 to 1997. One Spore-killer type was also found in a French strain dating from 1937. All other isolates found so far are sensitive to spore killing. All seven Spore killer types differ in the percentage of asci that show killing and in their mutual interactions. Interactions among Spore killer types showed either mutual resistance or dominant epistasis. Most killer elements could be assigned to linkage group III but are not tightly linked to the centromere.