Functional Characterization of AoFlbB and AoFlbD Reveals Their Roles in Sporulation and Trap Formation in Arthrobotrys oligospora

Sporulation is essential for asexual reproduction and dispersal in filamentous fungi, but the roles of upstream sporulation regulators in nematode-trapping (NT) fungi remain poorly understood. In this study, we characterized two Flb homologs, AoFlbB and AoFlbD, in Arthrobotrys oligospora. AoFlbB contains a bZIP-type domain, whereas AoFlbD contains a Myb-like DNA-binding domain, and both proteins are conserved among NT fungi. Deleting AoflbB or AoflbD had little effect on hyphal growth but markedly reduced spore production and altered spore germination, and real-time quantitative PCR analysis showed that the central sporulation regulators were significantly differently expressed, collectively indicating that AoFlbB and AoFlbD play positive roles in sporulation. Furthermore, both mutants produced more traps and showed enhanced nematode predation efficiency. The mutants also showed altered cell wall stress responses, hyphal cell length, nuclear number, endocytic trafficking, and metabolite profiles. The ΔAoflbB mutant showed irregular cell wall wrinkling, whereas the ΔAoflbD mutant displayed increased vesicle-like structures and accelerated FM4-64 uptake. Extracellular protease activity was comparable to the WT in ΔAoflbB but reduced in ΔAoflbD. Transcriptomic analysis of ΔAoflbD showed that the differentially expressed genes were significantly enriched in metabolism-related pathways. These findings indicate that AoFlbB and AoFlbD promote sporulation and contribute to the coordination of asexual reproduction and trap formation in A. oligospora.