A RID-like Cytosine Methyltransferase Homologue is essential for sexual development in Podospora anserina

Hélène Timpano ² Pierre Grognet ¹ Florian Carlier ¹ Jinane Ait Benkhali ¹ Véronique Berteaux-Lecellier ⁴ Robert Debuchy ¹ Frédérique Bidard ³ Fabienne Malagnac fabienne.malagnac@u-psud.fr ¹

¹Institute for Integrative Biology of the Cell, Université Paris Sud, Orsay, France
²Institute for the Diversity, Ecology and Evolution, Université Paris Sud, Orsay, France
³Biotechnologie, IFP Energies Nouvelles, Rueil-Malmaison, France
⁴UMR9220 Ecologie Marine Tropicale dans les Océans Pacifique et Indien, Institut d' Ecologie et Environnement, Noumea, France

In filamentous fungi, the Repeat Induced Point mutation (RIP) system, which is active during sexual reproduction, introduces G/C to A/T mutations within the repeats thus creating AT-rich regions. Originally described in Neurospora crassa, the presence of active RIP has then been evidenced in various other filamentous fungi, including P. anserina. To date the RID gene (RIP deficient), encoding a DNA methyltransferase-like protein, is the only one that has been demonstrated to be essential for RIP. But while RIP is conserved among filamentous fungi, DNA methylation does not seem so. Indeed, no significant levels of methylcytosines have been detected so far in the genomes of P. anserina and the chromatin status of their RIPped loci is still unknown.

To gain insight into PaRID function, we constructed a knocked-out ΔPaRid defective mutant. Remarkably, in contrast to N. crassa RID defective mutant, crosses involving P. anserina ΔPaRid mutants are sterile. We have shown that although gametes are readily formed and fertilization occurs in a ΔPaRid mutant background, the sexual development is blocked just before the individualization of dikaryotic cells. Conversely, knockout of the PaDim2 gene, encoding a second putative DNA methyltransferase related to N. crassa DIM-2, had no effect on Podospora’s complete life cycle. Complementation of the ΔPaRid mutant with ectopic alleles of PaRid, including GFP-tagged, point-mutated, inter-specific and chimeric alleles, demonstrated that the catalytic motif of the putative PaRID methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is tightly regulated in both space and time. Finally, a microarray transcriptomic analysis of the ΔPaRid mutant highlighted the genetic network differentially regulated in the absence of this putative DNA methyltransferase. Altogether, this study sheds a new light on sexual development regulation in a heterothallic model ascomycete.