The phospholipid flippase DNF-4 is implicated in membrane trafficking and hyphal development in Neurospora crassa

Phospholipid flippases (P4-ATPases) are central to the establishment of membrane lipid asymmetry, a property underlying membrane trafficking and polarized growth in eukaryotic cells. However, the specific contribution of individual flippases to hyphal morphogenesis in filamentous fungi remains poorly defined. Here, we characterize DNF-4, a putative P4-ATPase in Neurospora crassa with high sequence identity to the essential flippase Neo1 of Saccharomyces cerevisiae. Using endogenous tagging and live-cell imaging, we show that DNF-4 localizes to highly dynamic punctate structures associated with endoplasmic reticulum- and Golgi-related compartments, supported by quantitative co-localization analyses with the ER marker CSE-7-mChFP and the Golgi-associated Rab GTPase YPT-1-mChFP. FRAP experiments revealed partial fluorescence recovery, indicating dynamic exchange of DNF-4-associated compartments. These structures undergo bidirectional movement along the hypha, and their motility is strongly dependent on an intact microtubule cytoskeleton. Deletion of dnf-4 results in pronounced defects in hyphal growth and development, including reduced hyphal elongation, decreased biomass accumulation, smaller conidia, and a severe reduction in conidiation. These defects are accompanied by increased branching frequency, abnormal hyphal morphology, and altered Spitzenkörper positioning and dynamics, indicating impaired coordination of polarized growth. Our results demonstrate that DNF-4 contributes to membrane trafficking processes required for the maintenance of hyphal polarity and normal developmental progression in N. crassa. These findings provide new evidence that P4-ATPases play an important role in the spatial organization of membrane trafficking pathways underlying fungal morphogenesis.