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We cloned the pheromone precursor genes of Podospora anserina in order to elucidate their role in the biology of this fungus. The mfp gene encodes a 24-amino-acid polypeptide finished by the CAAX motif, characteristic of fungal lipopeptide pheromone precursors similar to the a-factor precursor of Saccharomyces cerevisiae. The mfm gene encodes a 221-amino-acid polypeptide, which is related to the S. cerevisiae alpha-factor precursor and contains two 13-residue repeats assumed to correspond to the mature pheromone. We deleted the mfp and mfm coding sequence by gene replacement. The mutations specifically affect male fertility, without impairing female fertility and vegetative growth. The male defect is mating type specific: the mat+ Deltamfp and mat- Deltamfm mutants produce male cells inactive in fertilization whereas the mat- Deltamfp and mat+ Deltamfm mutants show normal male fertility. Genetic data indicate that both mfp and mfm are transcribed at a low level in mat+ and mat- vegetative hyphae. Northern-blot analysis shows that their transcription is induced by the mating types in microconidia (mfp by mat+ and mfm by mat-). We managed to cross Deltamfp Deltamfm strains of opposite mating type, by complementation and transient expression of the pheromone precursor gene to trigger fertilization. These crosses were fertile, demonstrating that once fertilization occurs, the pheromone precursor genes are unnecessary for the completion of the sexual cycle. Finally, we show that the constitutively transcribed gpd::mfm and gpd::mfp constructs are repressed at a posttranscriptional level by the noncognate mating type.

The Function of the Coding Sequences for the Putative Pheromone Precursors in Podospora anserina Is Restricted to Fertilization