GSNOR plays roles in growth, pathogenicity, and stress resistance by modulating mitochondrial protein COX6B S-nitrosylation in <i>Colletotrichum</i> gloeosporioides

Nitric oxide (NO) serves as a versatile signaling molecule governing diverse biological processes, primarily through post-translational modifications such as S-nitrosylation. The enzyme glutathione S-nitrosoglutathione reductase (GSNOR) plays a central role in NO homeostasis by modulating cellular levels of S-nitrosoglutathione (GSNO), thereby controlling protein S-nitrosylation dynamics. However, the functional significance of GSNOR in fungal pathogenicity remains insufficiently characterized. In this study, we investigated the function of CgGSNOR in the phytopathogenic fungus <i>Colletotrichum gloeosporioides</i>. Deletion of <i>CgGSNOR</i> disrupted nitrosative homeostasis, leading to elevated NO accumulation, increased protein S-nitrosylation levels, and mitochondrial dysfunction as evidenced by reduced ATP production and altered ROS levels. Proteomic and structural analyses identified cytochrome c oxidase subunit 6B (CgCOX6B) as a key target of S-nitrosylation. Functional characterization revealed that CgCOX6B is essential for appressorial turgor maintenance and fungal pathogenicity. Site-directed mutagenesis demonstrated that three conserved cysteine residues (Cys42, Cys62, and Cys73) are critical for CgCOX6B function and are susceptible to S-nitrosylation-induced disruption. Notably, the <i>CgCOX6B</i> knockout strain exhibited increased sensitivity to Iprodione, a widely used fungicide, and this sensitivity was further amplified by NO donor treatment. Together, our findings uncover a GSNOR-dependent redox regulatory axis that links NO signaling, mitochondrial function, and fungal pathogenicity, offering potential targets for antifungal strategies via manipulation of NO signaling networks.IMPORTANCE<i>Colletotrichum gloeosporioides</i> is a globally significant fungal pathogen responsible for anthracnose diseases, causing losses across a wide range of crops. Although nitric oxide (NO) signaling and its post-translational regulatory mechanism, S-nitrosylation, are known to play pivotal roles in fungal biology, their specific contributions to pathogenicity remain poorly characterized. This study identifies glutathione S-nitrosoglutathione reductase (GSNOR) as a critical regulator of NO homeostasis in <i>C. gloeosporioides</i> and demonstrates its critical role in regulating fungal growth, conidiation, and pathogenicity. We uncover cytochrome c oxidase subunit 6B (COX6B) as a key target of S-nitrosylation, required for fungal energy metabolism, host infection, and resistance to fungicides. Furthermore, we reveal that exogenous NO supplementation using sodium nitroprusside synergistically enhances the antifungal activity of Iprodione. These findings advance our understanding of redox regulation in fungal pathogenesis and highlight GSNOR and COX6B as promising molecular targets for developing antifungal approaches to reduce crop losses.