Perithecium morphogenesis in Neurospora crassa and Sordaria macrospora

Multicellular development in fungi is fundamentally different from that of animals or plants. In filamentous fungi, multicellular structures are formed by aggregation and adhesion of hyphae, followed by septation and specialisation of hyphal compartments within the aggregate. The perithecium, a flask-shaped sexual fruitbody produced by both Neurospora crassa and Sordaria macrospora, provides a model system in which to study fungal multicellular development. This study presents a detailed description of the morphological stages of perithecial morphogenesis in N. crassa and S. macrospora and its early stages, the ascogonial and protoperithecial stages, using a range of microscopical techniques. Details of the development of several mutants impaired in perithecial development are described, including: gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; and three mutants pro22, pro40 and pro41 of S. macrospora, and their corresponding gene-deletions in N. crassa. The results confirm that all three MAP kinase cascades are required for sexual development. However, only the pheromone response and cell-wall integrity MAP kinase pathways, but not the osmoregulatory MAP kinase pathway, are essential for hyphal cell fusion. Evidence of cell fusion-related processes, regulated through MAP kinase signalling, have been identified as novel features important for the construction of fertilisable protoperithecia. These cell-fusion related processes include extracellular matrix deposition, hyphal attachment and envelopment. A novel phenotype of S. macrospora with defective ascogonial septation is presented. This pro22 mutant also has impaired hyphal cell fusion and produces only small, defective protoperithecia. The pro22 gene encodes a protein that is highly conserved throughout eukaryotes. Live-cell imaging revealed that this PRO22 protein is localised in the dynamic tubular and vesicular vacuolar-network of the colony periphery and in ascogonia. PRO22 is absent from the large spherical vacuoles in the vegetative hyphae of the sub-peripheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar-network. Furthermore, the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development