Down-Regulation of AGO2 Expression in Nicotiana benthamiana Does Not Compromise Normal Development but Interferes with Antiviral Silencing of a Tombusvirus

ARGONAUTE proteins (AGOs) in plants are thought to be important for antiviral RNA silencing by forming the siRNA-programmed catalytic slicer component of the RNA-induced silencing complex that sequence-specifically cleaves viral RNA. Select AGOs in Arabidopsis are known to contribute to antiviral silencing, but little is known about possible antiviral AGOs in other plants. We have reported that Tobacco rattle virus (TRV) vector mediated virus-induced gene silencing (VIGS) of Nicotiana benthamiana AGO2 (NbAGO2) compromises the ability of this plant to effectively mount an antiviral silencing defense response against Tomato bushy stunt virus (TBSV). To eliminate the contribution of possible interactive effects between TRV and TBSV, we wished to determine if constitutive down-regulation of NbAGO2 affected the anti-TBSV silencing response similar to what occurred with VIGS, and whether such knock-down had any noticeable developmental effects. For this purpose, we employed dsRNA hairpin technology to reduce NbAGO2 expression in N. benthamiana, as verified with RT-PCR. In absence of biotic stress during growth, the hairpin transgenic plants did not exhibit a phenotype that deviated from control plants. Inoculation of control plants with a GFP expressing and P19-suppressor deficient TBSV variant resulted in little or rapidly declining GFP levels due to antiviral silencing whereas GFP accumulation remained high in plants expressing the NbAGO2 hairpin. These findings provide evidence that while NbAGO2 in N. benthamiana is not essential for development under normal growing conditions, it is crucial for the antiviral response towards TBSV.