GUN Mutants: New Weapons To Unravel Ascospore Germination Regulation in the Model Fungus Podospora anserina

The regulation of ascospore germination in filamentous fungi has been poorly investigated so far. To unravel new genes involved in this regulation pathway, we conducted a genetic screen in Podospora anserina, and we isolated 57 mutants affected in ascospore germination. In Podospora anserina as in many other Ascomycetes, ascospore germination is a regulated process that requires the 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 (Germination UNcontrolled) mutants. Here, we report on the characterization of the gun1(SG) (Spontaneous Germination) mutant. We show that gun1(SG) is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl coenzyme A between peroxisomes and mitochondria and which is required for appressorium development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among 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 together with the PaMpk2 MAPK pathway upstream of the PaNox2/PaPls1 complex in the regulation of ascospore germination. In addition, we show that GUN1 plays a role in appressorium functioning. The pivotal role of GUN1, the ortholog of Pth2, in ascospore germination and in appressorium functioning reinforces the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. Furthermore, 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.IMPORTANCE The regulation of ascospore germination in filamentous fungi has been poorly investigated so far. To unravel new genes involved in this regulation pathway, we conducted a genetic screen in Podospora anserina, and we isolated 57 mutants affected in ascospore germination. Here, we describe the Germination UNcontrolled One (gun1(SG)) mutant, and we characterize the gene affected. GUN1 is a peroxisomal/mitochondrial carnitine-acetyltransferase required for acetyl coenzyme A shuttling between both organelles, and we show that GUN1 is a pleiotropic gene also involved in appressorium functioning similarly to its ortholog, the pathogenesis factor Pth2, in the plant pathogen Magnaporthe oryzae. Given the similarities in the regulation of appressorium development and ascospore germination, we speculate that discovering new genes controlling ascospore germination in P. anserina may lead to the discovery of new pathogenesis factors in pathogenic fungi. The characterization of GUN1, the ortholog of M. oryzae Pth2, represents a proof of concept.

Automated summary

By conducting a genetic screen in Podospora anserina, researchers identified a new gene, gun1, involved in the regulation of ascospore germination. The gun1 mutant showed uncontrolled germination and also played a role in ascospore development. This study provides new insights into the regulation of ascospore germination in filamentous fungi.