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Abstract Fungal species are typically either fully self-fertile or self-sterile, but some filamentous ascomycetes can commonly transition from self-fertility to self-sterility through unidirectional mating-type switching. In these fungi, the structure of the mating-type ( MAT1 ) locus governs sexual behaviour: MAT-2 self-fertile individuals retain both MAT1-1 and MAT1-2 genes, while MAT-1 self-sterile isolates lose MAT1-2 genes during switching. A third type of isolate morphology also occurs under laboratory conditions: these are self-sterile isolates which retain both MAT1-1 and MAT1-2 , but are unable to switch mating type. These are commonly referred to as MAT-2 self-sterile isolates. Two of the mating-type ( MAT ) genes, one of which is deleted during switching, encode transcription factors known to regulate not only the sexual cycle but also genes unrelated to mating. To test how MAT1 structural variations affects gene expression, we studied Ceratocystis albifundus , a species that switches mating type. To minimise variability caused by intraspecific genetic differences, two self-sterile isolates (MAT-1 and MAT-2 self-steriles) were derived from the same MAT-2 self-fertile parent, making all three isolates genetically identical except at the MAT1 locus. Comparative transcriptomic analyses revealed that the MAT-2 self-fertile, MAT-1 self-sterile and MAT-2 self-sterile isolates all exhibited distinct expression patterns, including differences in MAT genes, the pheromone–receptor pathway, and other genes not directly linked to mating. The results show that MAT1 locus structure influences gene expression more broadly than those only related to the sexual cycle.