Podospora anserina

One model does not fit all

Fruiting bodies of Podospora anserina. Sexual reproduction occurs when the culture medium is used up: fertilization is then followed by the formation of fruiting bodies, the perithecia. Karyogamy (fusion of the male and female nuclei) takes place within these specialized protective structures, followed by meiosis and the formation of asci, each of which contains four binucleate ascospores. About four days after fertilization, the ascospores are ejected to the exterior of the perithecium.

The filamentous ascomycete fungus Podospora anserina is a model organism that is used for the genetic and molecular study of several biological processes. The vast phylum Ascomycetes includes species with very diverse lifestyles; several are cultivated in the laboratory and studied as models by large groups of scientists. Among the model species for which the genomes have already been sequenced, Neurospora crassa is the closest to Podospora anserina. These two fungi belong to the same order, the Sordariales, within the class Sordariomycetes. The Neurospora and Podospora lines are thought to have diverged at least 75 million years ago.

Neurospora crassa became famous as a model species because of its role in the birth of molecular biology : the demonstration of the famous “one gene-one enzyme” relationship by George W. Beadle and Edward Tatum in 1940 was based on the genetics of Neurospora. However, the Podospora anserina model was preferred on this side of the Atlantic, due to the pioneering work of Georges Rizet. Initiated by these studies, a rich tradition of microbial genetics grew up in France around Podospora and other filamentous fungi (Ascobolus immersus, Sordaria macrospora, etc.), especially at the Faculty of Sciences at Orsay.

Podospora anserina is found in nature on herbivore dung. This species undergoes sexual reproduction, with a haplobiontic cycle (the diploid nucleus formed by karyogamy immediately undergoes meiosis) which lasts about one week. In this haploid organism, mutations can therefore be detected easily and rapidly. The asci contain 4 spores, each with 2 haploid nuclei.

The study of Podospora anserina shows that we would be wrong to limit ourselves to a single model such as Neurospora crassa. Despite the fact that they are relatively closely related, the two species present major differences. Podospora provides access to different biological phenomena than Neurospora. In particular, the hyphae of Podospora anserina undergo a phenomenon of senescence which has established this fungus as a model for the study of the mechanisms of aging for decades. In Neurospora crassa, on the contrary, senescence is not systematically observed. Furthermore, the phenomena of gene extinction, which are especially efficient in Neurospora crassa are not present - or are present with less efficiency - in Podospora anserina, which makes it possible to develop technologies in Podospora which could not succeed in Neurospora.

Biological processes studied in Podospora anserina

Fundamental biological processes studied in Podospora anserina are mainly:

• henomena of cell degeneration, and the role of non-conventional infectious agents in these syndromes and in senescence.
• The control of the accuracy of translation in the cytosol, which is implicated in senescence and which seems to play an important role in the stability of these infectious agents.
• Instability of the mitochondrial genome, which is a cause of mitochondrial dysfunction - a feature which is found in a number of human myopathies -, and its association with the process of senescence, perhaps through the lack of accuracy of the translation (mitochondrial proteins incorrectly translated in the cytosol would cause the damage).
• The relationship between senescence and respiratory function which is based in the mitochondria. This is a pattern which has been found in the study of aging in other species.
• A phenomenon of cell death which is called vegetative incompatibility, and which follows the fusion of somatic cells of individuals who are genetically incompatible.
• The presence of a prion, which is capable of aggregation to form amyloid fibers, similar to those found in spongiform encephalopathies and Creutzfeldt-Jakob disease in humans, and which may have a role in vegetative incompatibility.
• The control of development during the sexual cycle, and cellular differentiation.
• The evolution of the various systems of sexual reproduction and the evolution of multicellularity.

Reasons for sequencing

A large number of genes implicated in these various processes have been localized, because Podospora anserina lends itself well to genetic analysis. However, only a few of these genes have been characterized at the molecular level to date. The sequencing of the genome of Podospora anserina will accelerate this research in the following manner:

• By facilitating the cloning of genes. With the availability of the complete sequence, as long as one knows what one is looking for, it will be easier to clone a fragment of sequence by PCR or to find it in the libraries used for sequencing which are now available. In the opposite situation, the genomic sequence will also be useful: at the present time, cloning of a Podospora gene by complementation of a mutant can take up to a year; this time span will be reduced by a factor of 10 by a positional cloning procedure. It is already known that Podospora anserina contains micro- and minisatellites which are polymorphic from one population to another and could therefore be used as genetic markers. The candidate genes localized in intervals between some of these markers could then be tested by functional complementation after transformation.
• By improving the annotation of the sequence of the genome of Neurospora crassa, and more generally, genomic sequences of other filamentous fungi. On the other hand, the genomic sequence of Neurospora crassa will also facilitate the annotation of Podospora anserina.
• By initiating comparative studies on the genomes of filamentous fungi, and then extending the comparisons to yeast genomes, and beyond, to those of metazoans (animals and fungi are close parents among eukaryotes) and plants (for which the lifestyles are similar in some aspects to those of fungi). It is important to emphasize here that filamentous fungi have more proteins which are homologous to human proteins than yeast have.
• To envisage the development of DNA chips containing the ensemble of Podospora anserina genes for the study of gene expression profiles associated with the various cellular phenomena which have been characterized in this fungus.

The Podospora anserina genome contains 7 chromosomes. Its size is about 34 Mb as estimated from pulsed-field electrophoresis. This genome is therefore smaller than that of Neurospora crassa, which is about 40 Mb and was sequenced in 2003 with 10X coverage. Other filamentous fungi sequenced to date include Magnaporthe grisea - another sordariomycete - and other species less closely related to Podospora: Fusarium graminearus, Aspergillus nidulans, Aspergillus fumigatus, Phanerochaete chrysosporium and Coprinus cinereus. The genomes of even more fungus species, mainly plant or human pathogens, are in the process of being sequenced.

History of the project

Beginning in September 2000, Genoscope became involved in a pilot project on the centromeric region of chromosome V of Podospora anserina. This project had been submitted at the end of 1999 by four laboratories working on this fungus. Their goal was to study the feasibility of a whole genome sequencing project. At the end of the pilot project in January 2002 (see the references of the article), two contigs with a cumulative size of 480 kb were obtained; they made it possible to establish a value of 50% as the GC percentage of Podospora, and to demonstrate the rarity of repeated sequences. This data was favorable for the perspective of a whole genome shotgun approach. Furthermore, this pilot project also revealed the compact nature of the Podospora genome (only about 1 kb between genes) and the small average size of introns (60 bps), which will facilitate the annotation procedure. The immediate benefit of the pilot project was the identification of two genes implicated in cell degeneration phenomena, and of 160 other genes from which several consensus sequences were defined for the identification of introns and coding sequences. Finally, these sequences should help in the identification of a filamentous fungus centromere, which will be useful for the construction of a non-integrated vector for this type of organism.

The whole genome sequencing project began in 2003 in the framework of a “Large-Scale Sequencing” grant program from the Ministry of Research and New Technologies. Initial funding was for the production of sequences at 3X coverage at Genoscope. The project was then extended to random shotgun sequencing of the Podospora anserina genome at 10X coverage. This new goal was advocated by the Scientific Council of Genoscope and a consortium of five French groups and a Dutch group (see Collaboration), coordinated by the “Genetic control of cellular degenerative processes” group of the Institut de Génétique et Microbiologie (UMR CNRS 8621) at the University of Orsay-Paris Sud. The project has also received support from the Institut Fédératif de Recherche “Genome” at the Orsay - Gif-sur-Yvette Centre.

In addition to finishing the genome sequence, the “Podospora” consortium will perform the annotation. Extrapolation from the sequences annotated in the pilot project have indicated the presence of about 11 000 genes in the sequence of this fungus. The annotation process will use sequences from cDNA clones of Podospora anserina which were produced at Genoscope. It will also benefit from the comparison with the genome of Neurospora crassa. This fungus seems to be situated at the proper evolutionary distance from Podospora for the two genomes to be compared by the procedure Exofish which was developed at Genoscope: more than 95% of the genes which have already been found in Podospora have a homolog in Neurospora, whereas introns and intergenic sequences are not conserved. The evolutionary conserved regions revealed by Exofish will therefore correspond to exons with high sensitivity and specificity.

Philippe Silar et al. (2003) Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing, Fungal Genetics and Biology 39 n°3, pages 250-263.

The groups forming the “Podospora” consortium are:

• The “Genetic control of cellular degenerative processes” group of the Institute of Genetics and Microbiology (IGM, UMR CNRS 8621) at the Orsay University.
• The “Sexual reproduction, translation and differentiation” group of the IGM at the Orsay university.
• The “Senescence and longevity in fungusP. anserina” group of the Centre de Génétique Moléculaire (CGM) at CNRS in Gif-sur-Yvette.
• The fungal molecular genetics laboratory of the Institut de Biochimie et Génétique Cellulaires (IBGC) at the university Bordeaux II in Talence.
• The “Replication and expression of eucaryotic et retroviral genomes” (REGER) laboratory at the university Bordeaux II.
• The laboratory of genetics of the Wageningen university.

The informatics aspects of the annotation work will be directed by Olivier Lespinet, who works in the “Molecular evolution and genomics” group of the IGM at the University of Orsay.