The GUN mutants: new weapons to unravel ascospore germination regulation in the model fungus Podospora anserina

In Podospora anserina as in many other ascomycetes, ascospore germination is a regulated process that requires breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN mutants. In this paper, we report on the characterization of GUN1SG. We show that GUN1SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a Carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl-CoA between peroxisomes and mitochondria and which is required for appressorium-development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among the Fungi, and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 upstream of the PaMpk2 MAPK pathway and the PaNox2/PaPls1 complex in the regulation of ascospore germination. The identification of GUN1, the ortholog of Pth2, in ascospore germination, strengthens the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. In addition, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina.