Genomic conflicts in Podospora anserina = Genomische conflicten in Podospora anserina

This thesis deals with genomic conflicts raised by selfish elements in the ascomycete fungus Podospora anserina .Genomic conflicts arise when the effects of the selfish elements are opposite to the interests of the other parts of the genome. Two types of selfish elements are studied as well as certain characteristics of Podospora involved in the population dynamics of these elements, such as vegetative and sexual incompatibility, senescence and outcrossing.The natural habitat of Podospora anserina is dung of herbivores where it has an optimum growth temperature of 27 °C. The fungus can only reproduce sexually and the ascospores are the products of meiosis as well as the next generation of the fungus. Perithecia or fruiting bodies contain asci with four linearly arranged ascospores, which provide unique opportunities to analyse abnormal segregation and makes this fungus one of the genetic model organisms. Most ascospores are capable of completing the lifecycle of the fungus, as they contain two nuclei, each with one of the two mating types. This fungal trait is called pseudo or secondairy homothallism, and it allows sexual offspring to be produced by either selfing or outcrossing. Sometimes smaller single mating type ascospores are formed containing one nucleus and less cytoplasmic content and mitochondria. Colonies from these spores must outcross with another isolate to produce offspring. The fungal isolates used in this thesis were sampled from dung around Wageningen, theNetherlandsduring 1990-1997, but also some older French isolates dating from 1937 were used.The first selfish elements studied in this thesis are linear plasmids with homology to pAL2-1, a 8.3 kb plasmid previously found in this fungus. Linear plasmids are parasitic autonomous replicating genetic elements. In filamentous fungi they reside in the mitochondria. Most plasmids are cryptic or have a negative fitness effect on the host. However the pAL2 plasmid found in P. anserina has been associated with a longevity phenotype, though recently also negative effects were found. Homologous linear plasmids and plasmid families were present in 21 percent of the population and detected over several years. Half of the plasmid containing isolates possesseda single plasmid and the other half a plasmid family consisting of multiple plasmid copies. The lifespan of the isolates was generally uncorrelated with the presence of the plasmid. One isolate showed an increased lifespan, but noinserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity inducing pAL2-1 plasmid. We have looked at the dynamics of plasmid transmission for these plasmids.Vertical transfer of the plasmids to the ascospores occurs only via the maternal line. This transfer is inefficient as up to 40% lose all plasmids in an outcrossing situation and up to 20% retain only the basic plasmid family member in presence of a plasmid family. No difference in efficiency of plasmid transfer to the dikaryotic and the smaller monokaryotic ascospores was detected. Plasmid transmission was also foundindependent of the genetic background of the strains.Loss of plasmids via the sexual route is compensated by horizontal transmission of the plasmid through hyphal contact between different isolates. Horizontal transfer occurs efficiently in both vegetative compatible and incompatible situations. Vegetative incompatibility can be macroscopically observed as a 'barrage', a contact zone of lysed hyphal cells. Vegetative incompatibility is thought to have evolved as a mechanism to hamper spread of mobile selfish elements and parasites. Our experiments show that vegetative incompatibility is not a perfect barrier against this type of selfish elements.The other selfish elements studied in this thesis are meiotic drive factors or segregation distorters. Segregation distorters are transmitted into the progeny in excess of the fair Mendelian proportion of 50 %, by actively destructing the alternative allele. Genomic conflicts arise by hitchhiking of genes with deleterious fitness effects. Meiotic drive in Podospora ischaracterized by the abortion of two of the four spores in the ascus.Seven different groups of meiotic drive elements or Spore killer types were identified and characterized. Among 99 isolates from nature, six of these meiotic drive elements occurred in our local population. All drive elements comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands and most elements can be retrieved over the years. Spore-killer type Psk-7 was also present in a French strain dating from 1937 and exists for more than 60 years. No resistance to meiotic drive was observed and all other isolates found so far are sensitive to spore killing. Each type of Spore killer differs in the percentage of asci that show killing, ranging from 50 to 95% two-spored asci, and in their mutual interactions. The aborted ascospores quickly degrade after spore wall formation, except for the Psk-3 group where they remain visible as tiny shriveled ascospores, indicating different abortion factors or timing for killing. The Psk-3 group also shows a variable percentage of two-spored asci within each perithecium. Spore-killer interactions show either mutual resistance ( i.e. no abortion is found if an allele of either Spore killer is present) or dominant epistasis ( i.e. one killer acts as a sensitive type). Genetic mapping could assign most Spore-killer types to linkage group III where they are not tightly linked to the centromere.Several possible models that explain the spore killing mechanism in Podospora anserina were examined. Repeatedbackcrossing of Spore killers to the same sensitive isolate produces strains with the same genetic background. Spore killers that belong to the same killer type or show mutual resistance become vegetative compatible to each other during backcrosses. On the other hand, Spore killers that show dominant epistasis, as well as the sensitive strain remained vegetative incompatible. This suggests a common mechanism for spore killing, possibly related to vegetative incompatibility, although the precise genetic nature of the correlation is not yet clear.The Podospora genome was screened for homologues of genes known to be involved in silencing in fungi. Genes were found for all silencing mechanisms (RIP, MSUD, Quelling) known in the related fungus Neurospora . However, the possible role of silencing by methylation of genes during the killing process was excluded by experiments using the drug 5-azacytidine, which both removes methylation and prevents de-novo methylation. No effect of the 5-azacytidine treatments was found on spore-killing frequency for all Spore-killer types. Also the consequences of formation of dikaryotic ascospores for expression of spore killing were examined. Crosses were used of Spore killers with a marker that increases the number of monokaryotic spores to up to eight. Spore-killer types Psk-2 , Psk-3 (Wa27) and Psk-4 produced some asci containing more than four spores in these crosses. In such asci sensitive nuclei were able to survive and resist the meiotic drive system, indicating incomplete penetrance of the spore-killing mechanism. Spore killing in the other killer types ( Psk-1, Psk-5, Psk-6 and Psk-7 ) were full penetrant; only asci with four or less spores could be detected. Here the killing mechanism works similar on all asci. Furthermore the effect of low temperatures (22 ºC) on spore killing was tested in this Chapter. Psk-2 dramatically decreased the percentage of killing at this temperature to almost zero. Other Spore-killer types were not affected by temperature. Based on all characteristics and interactions of the Spore killers we propose that spore killing in Podospora may be an example of post-segregational killing due to Toxin-Antitoxin mechanisms. A Spore Killer produces both a persisting stable toxin and a less stable antitoxin. In ascospores where the Spore Killer is absent the antitoxin disappears more quickly than the toxin, leading to abortion of the spores. Variable killing percentages can be explained by the strict balance between toxin and antitoxin and the timing of shutdown of the genes involved.Meiotic drive is only possible in an outcrossing situation. Podospora is in principle capable of both outcrossing and selfing. However to what extent the fungus outcrosses in nature is unknown. The likelihood of outcrossing was assessed for the secondary homothallic ascomycete Podospora anserina. We examined the extent of vegetative en sexual compatibility between wild type strains. The number of vegetative compatibility groups (VCG's) in the population was estimated based on the incompatibility reactions between isolates in our survey using accumulation curve extrapolation and the non-parametric Chao1 formula. The estimated number of VCG's compared to the maximum number of VCG's based on the currently known vegetative incompatibility genes suggest regular outcrossing in this fungus. Also the difference in sexual compatibility reactions of mating types of the same isolate assumes outcrossing takes place. Options for outcrossing in P. anserina were experimentally verified . Both single mating type monokaryotic and dikaryotic double mating type mycelial cultures proved capable of outcrossing, showing no preference for either genotype. This indicates that fertilization by selfing and outcrossing uses the same pathway. Outcrossing percentages between 1-5 percent were found in unmanipulated natural situations of ascospores on dung. The number of monokaryotic ascospores found in Spore-killer ( Psk ) strains was significantly higher than in other isolates, showing an enhancement to outcrossing in these strains.The findings in this thesis contribute to the understanding of the population dynamics and evolution of two types of selfish elements, linear plasmids and segregation distorters, in the ascomycete fungus Podospora anserina. Furthermore it increases the knowledge on genomic conflicts caused by these types of selfish elements in general