Background: The ubiquitin–proteasome system plays a critical role in plant antiviral defense, with HECT-type E3 ubiquitin ligases serving as key regulators of protein turnover. To explore the potential involvement of the HECT gene family in host resistance against tomato yellow leaf curl virus (TYLCV), a comprehensive analysis was conducted in Nicotiana benthamiana. Methods: In this study, the HECT gene family in N. benthamiana was systematically investigated using a genome-wide bioinformatic analysis. The potential roles of these genes in the response to TYLCV infection were further examined using a virus-induced gene silencing (VIGS) technique. Results: Using a Hidden Markov Model approach, 18 NbHECT genes were identified that phylogenetically clustered into four subfamilies with distinct structural features. Chromosomal location and synteny analyses indicated that these genes were unevenly distributed across 11 chromosomes, with 10 instances of segmental duplication identified. Tissue-specific expression profiling demonstrated that 17 NbHECTs were constitutively expressed, with Group III members showing the highest expression in reproductive tissues. Following TYLCV infection, NbHECT6 was significantly downregulated while NbHECT13 was upregulated in both inoculated and systemic leaves. Functional validation through the VIGS approach revealed that suppression of NbHECT6 and NbHECT13 increased host susceptibility, as evidenced by exacerbated symptom severity and enhanced viral DNA accumulation compared to controls. Conclusions: These findings establish NbHECT6 and NbHECT13 as critical components of the plant antiviral response, providing new insights into ubiquitin-mediated defense mechanisms against geminiviruses.

Copper (Cu) contamination in aquatic environments induces oxidative stress and structural damage to crustaceans. This study investigated the protective effects and associated mechanisms of exogenous melatonin (MT) against Cu-induced toxicity in Procambarus clarkii using integrated physiological, histopathological, proteomic, and molecular analyses. MT supplementation enhanced antioxidant defense by elevating SOD, CAT, and T-AOC activities, while reducing MDA accumulation, with peak effects observed at 24 h. MT also restored endogenous melatonin levels and regulated phosphatase activity, thereby maintaining immune and metabolic homeostasis. Histopathology showed reduced hepatopancreatic damage, characterized by reduced epithelial vacuolization and preserved basement membrane integrity. Proteomics suggested that MT modulates a multilayered network associated with detoxification, redox balance, and cellular homeostasis. Pathway enrichment showed that Cu exposure dysregulated proteins involved in mitochondrial biogenesis, ABC transporters, membrane trafficking, and apoptosis. MT administration partially counteracted these alterations and was associated with the regulation of glutathione metabolism, as well as reduced enrichment of lysosome- and apoptosis-related pathways. Quantitative RT-PCR results were consistent with the proteomic data. Overall, MT partially alleviated Cu-induced toxicity and was associated with enhanced antioxidant defense, improved cellular homeostasis, and metabolic regulation. Our study provides new molecular insights and suggests its potential application for mitigating metal toxicity in aquaculture.