Spatiotemporal compartmentalization of NADPH oxidase signaling coordinates cell fusion and predatory behavior in nematode-trapping fungi

. We demonstrate a distinct functional specialization between the two NADPH oxidases NoxA and NoxB, which are both activated by the regulatory subunit NoxR. The NoxB-NoxR complex was specifically active in trap cells, where it was required for producing the adhesive "glue" for prey capture. In contrast, NoxA localized to the plasma membrane of vegetative hyphae and was required for hyphal fusion and trap closure. NoxA was activated by the pulsatile recruitment of NoxR to the hyphal tip, suggesting oscillatory ROS production during hyphal expansion and hyphal fusion. Nox complexes initially generate superoxide, which is probably converted to more stable hydrogen peroxide (H₂O₂) and serves as a key spatial signal. Together, these findings reveal a remarkable division of labor between Nox isoforms, where spatial compartmentalization of ROS signaling drives fungal morphogenesis and predatory behavior. IMPORTANCE: Reactive oxygen species (ROS) are not only toxic byproducts of mitochondrial activity but also important signaling molecules in eukaryotes. To fulfill this signaling role, ROS production must be tightly regulated. NADPH oxidases are key contributors to controlled ROS generation, as they may be regulated at the transcriptional level, targeted to specific cellular locations, and activated by dedicated regulatory proteins. Here, we investigated the role of NADPH oxidases in the nematode-trapping fungus Arthrobotrys flagrans and show that two NADPH oxidases serve distinct roles: one is required for trap function, while the other mediates cell-to-cell communication and is required for hyphal fusion and trap closure. The latter finding suggests ROS, most likely H₂O₂, as a diffusible signaling molecule between communicating fungal cells.