GSNOR is essential for nitric oxide homeostasis and involved in aflatoxin biosynthesis and pathogenicity in Aspergillus flavus.

Nitric oxide (NO) is an important biological signaling molecule. S-nitrosoglutathione reductase (GSNOR), a master regulator of NO signaling, regulates various biological processes. However, little is known about the role of GSNOR in Aspergillus flavus. Here, we identified a gene encoding GSNOR in this aflatoxigenic fungus and demonstrated that GSNOR shows activity during the critical life cycle stages, including spore germination, hyphal growth, and conidiogenesis. We found that GSNOR plays a crucial role in NO homeostasis, as GSNOR deletion resulted in significantly elevated NO levels and heightened sensitivity to exogenous NO stress. GSNOR also participated in multiple biological processes in A. flavus; for that, GSNOR deletion impaired conidia germination, reduced growth, decreased conidiogenesis and sclerotial development, attenuated virulence on kernels, and notably decreased aflatoxin production. Furthermore, we demonstrated that GSNOR is important for reactive oxygen species (ROS) balance, as its deletion significantly elevated mycelial ROS levels and made the strain more sensitive to oxidative stress.IMPORTANCEAspergillus flavus is a notorious saprophytic filamentous fungus, with its production of carcinogenic aflatoxins posing serious threats to food safety and human health. Aflatoxin contamination prevention and control have long been a global challenge. In previous studies, we observed that nitric oxide (NO) significantly inhibits the aflatoxin production by A. flavus. This study further investigated the role of the key regulatory enzyme S-nitrosoglutathione reductase (GSNOR) in the NO signaling pathway. Our findings indicate that GSNOR is crucial for maintaining both NO homeostasis and reactive oxygen species (ROS) balance and plays an essential role in fungal development, pathogenicity, and aflatoxin biosynthesis. These results highlight the potential of targeting components in the NO signaling pathway, such as GSNOR, as a novel strategy for the early prevention of aflatoxin contamination in food.