Genome Sequencing-Assisted Identification of Mechanisms Responsible for Unidirectional Mating Type Switching in the Fungal Pathogen Sclerotinia trifoliorum

The necrotrophic fungal legume pathogen Sclerotinia trifoliorum exhibits ascospore dimorphism and unidirectional mating type switching - self-fertile strains (L mating type) derived from large ascospores produce both self-fertile (large-spores) and self-sterile (small-spores, S mating type) offsprings in a 4:4 ratio. Genome sequencing of an L mating type strain assisted identifying the mating type (MAT) locus of 14 kb DNA region. PCR amplification and DNA sequencing with 14 overlapping primer pairs allowed comparison of the MAT locus between L mating type and S mating type strains. Four main mating type genes (MAT1-1-1, MAT1-1-5, MAT1-2-1 and MAT1-2-4) were found in the L mating type. However, a 2891-bp region along with the entire MAT1-2-1 and MAT1-2-4 genes had been completely deleted from the MAT locus in the S mating type. Meanwhile, two copies of a 146-bp direct repeat motif flanking the deleted region were found in the self-fertile (S mating type) strain, but only one copy of this 146-bp motif (a part of the MAT1-1-1 gene) was present in the S mating type strain. The two direct repeats were believed to be responsible for the deletion through homologous recombination in meiosis. Tetrad analyses showed that all small ascospore-derived strains lacked the missing DNA between the two direct repeats that was found in all large ascospore-derived strains. In addition, heterokaryons at the MAT locus was observed in field isolates as well as in laboratory derived single-ascospore isolates, suggesting that such direct repeat-mediated deletion may also possibly occur during mitosis.