Genome-Wide Analysis of the HECT-Type E3 Ubiquitin Ligase Gene Family in Nicotiana benthamiana: Evidence Implicating NbHECT6 and NbHECT13 in the Response to Tomato Yellow Leaf Curl Virus Infection

<b>Background:</b> 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 <i>Nicotiana benthamiana</i>. <b>Methods:</b> In this study, the HECT gene family in <i>N. benthamiana</i> 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. <b>Results:</b> Using a Hidden Markov Model approach, 18 <i>NbHECT</i> 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 <i>NbHECTs</i> were constitutively expressed, with Group III members showing the highest expression in reproductive tissues. Following TYLCV infection, <i>NbHECT6</i> was significantly downregulated while <i>NbHECT13</i> was upregulated in both inoculated and systemic leaves. Functional validation through the VIGS approach revealed that suppression of <i>NbHECT6</i> and <i>NbHECT13</i> increased host susceptibility, as evidenced by exacerbated symptom severity and enhanced viral DNA accumulation compared to controls. <b>Conclusions:</b> These findings establish <i>NbHECT6</i> and <i>NbHECT13</i> as critical components of the plant antiviral response, providing new insights into ubiquitin-mediated defense mechanisms against geminiviruses.