Vanillin Activates HuTGA1-HuNPR1/5-1 Signaling to Enhance Postharvest Pitaya Resistance to Soft Rot

<i>Fusarium oxysporum</i>-induced soft rot severely threatens postharvest pitaya quality and storage life, and while vanillin shows promise in the disease management, its mechanisms for controlling pitaya decay remain incompletely understood. In this study, we systematically investigated the molecular mechanism by which vanillin inhibits soft rot in postharvest pitaya, employing physiological and biochemical characterization, bioinformatics analysis, and molecular biology techniques. Compared with control fruit on 10 d, vanillin treatment significantly reduced disease index and lesion area by 27.12% and 67.43%, respectively. Meanwhile, vanillin treatment delayed the degradation of total soluble solids (TSSs) and titratable acidity (TA) and promoted the accumulation of total phenolics and flavonoids. Additionally, vanillin enhanced the activities of defense-related enzymes, such as catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), <i>β</i>-1,3-glucanase (GLU), chitinase (CHI), peroxidase (POD) and polyphenol oxidase (PPO), and increased antioxidant capacity, as evidenced by increased DPPH radical scavenging capacity and ascorbic acid content. This resulted in reduced oxidative damage, as indicated by decreased levels of malondialdehyde (MDA), H₂O₂ and O₂^•-. Yeast one-hybrid (Y1H), dual-luciferase reporter (DLR) and subcellular localization revealed that HuTGA1, a nuclear-localized transcriptional activator, specifically bound to the as-1 cis-acting element and activated expression of <i>HuNPR1</i> and <i>HuNPR5-1</i>. Transient overexpression of <i>HuTGA1</i> reduced reactive oxygen species (ROS) accumulation and upregulated related genes. These findings suggest that vanillin treatment might enhance pitaya resistance by activating the HuTGA1-HuNPR signaling module, providing insights into the molecular mechanisms underlying vanillin-induced resistance.