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a b s t r a c tChitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkörper (Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2-GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/ csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5-GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages.

Highlights Chitin and β-1,3-glucan synthases are transported separately in chitosomes and macrovesicles. Chitin synthases occupy the core of the SPK; β-1,3-glucan synthases the outer layer. CHS-4 arrival to the SPK and septa is CSE-7 dependent. Rabs YPT-1 and YPT-31 localization at the SPK mimics that of chitosomes and macrovesicles. The exocyst acts as a tether between the SPK outer layer vesicles and the apical PM.

bMost models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H ؉ -translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H ؉ out of the cell, generating a large electrochemical gradient that supplies energy to H ؉ -coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 m) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex. One of the main features of filamentous fungi is their apical mode of growth. In filamentous fungi, cell wall growth and exocytosis are linked processes that involve the highly polarized traffic of cell wall-building secretory vesicles to apical areas, where they deliver proteins and lipids. One of the most widely accepted models for fungal growth, the vesicle supply center for fungal morphogenesis, postulates a unidirectional traffic of vesicles to the hyphal apex, where they aggregate temporarily at an apical structure, the Spitzenkörper, prior to fusion with the apical plasma membrane (PM) by the process of exocytosis (1). However, some PM proteins, such as the H ϩ -ATPases, essential for hyphal growth, have been previously predicted to be absent or inactive at the hyphal apex (2-4), suggesting the existence of vesicle delivery routes other than the above and independent of the Spitzenkörper that reach nonapical regions of the hyphal PM. H ϩ -ATPases are involved in pumping protons out of the cell, generating a large electrochemical gradient and supplying energy to H ϩ -coupled nutrient uptake systems (5). This electrochemical gradient has been studied in several fungal species by diverse methods, including vibrating probes, microelectrodes, and pH indicators. The results showed that current normally flows inward at the hyphal apical regions and flows outward at distal areas (3, 4, 6-9).PMA1, the H ϩ -ATPase-encoding gene in Saccharomyces cerevisiae (SCRG_01016 [10]), has a single homolog in Neurospora crassa (NCU01680 [11,12]). In S. cerevisiae, Pma1p is one of the most abundant proteins of the cell surface (25 to 50%), while in N. crassa, it constitutes about 5 to 10% of the...

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