A Polycistronic tRNA-amiRNA System Reveals the Antiviral Roles of NbAGO1a/1b/2 Against Soybean mosaic virus Infection

RNA interference (RNAi) is a crucial antiviral defense mechanism in plants, where Argonaute (AGO) proteins play a central role. However, the function of AGO proteins in the interaction between <i>Soybean mosaic virus</i> (SMV) and <i>Nicotiana benthamiana</i> remains unclear. In this study, SMV pathogenicity was confirmed using an SMV-GFP infectious clone, with typical symptoms and systemic GFP fluorescence observed 14 days post-inoculation. Real-time quantitative reverse transcription polymerase chain reaction analysis revealed dynamic regulation of multiple <i>NbAGO</i> genes upon infection. Notably, <i>NbAGO1a</i>, <i>NbAGO1b</i>, and <i>NbAGO2</i> were significantly upregulated and positively correlated with viral accumulation, suggesting their critical roles in antiviral defense. Based on these findings, these three genes were selected as targets for artificial microRNA (amiRNA) silencing. Three amiRNAs were designed for each gene using the <i>Arabidopsis</i> miR1596 backbone, with the most effective sequences exhibiting silencing efficiencies ranging from 75.2% to 98.1%. A polycistronic tRNA-amiRNA (PTA) cassette was constructed using Golden Gate cloning technology to simultaneously target all three genes. Co-infection assays indicated that the PTA cassette enhanced SMV accumulation more effectively than single amiRNAs, as evidenced by increased GFP fluorescence (49.1-60.5%) and pronounced leaf necrosis. The PTA system downregulated the expression of <i>NbAGO1a</i>, <i>NbAGO1b</i>, and <i>NbAGO2</i> by 18.4-26.7%. Furthermore, silencing <i>NbAGO2</i> alone resulted in severe necrosis, underscoring its essential role in this antiviral defense mechanism. This study elucidates the importance of <i>NbAGO1a</i>, <i>NbAGO1b</i>, and <i>NbAGO2</i> in antiviral immunity and demonstrates the utility of the PTA system for efficient multi-gene silencing, offering valuable insights for developing RNAi-based antiviral strategies.