RNA interference (RNAi) was established in Nicotiana benthamiana plants by introducing constructs containing a defective interfering (DI) sequence from Tomato bushy stunt virus (TBSV) in combination with a conserved sense-sequence from the target Grapevine fanleaf virus (GFLV). Silencing in plants was confirmed by Agrobacterium-mediated infiltration of a GFP-sensor containing the GFLV-derived target sequence. The transgene-induced RNAi led to silencing of the GFP-sensor and GFP fluorescence was absent post-infiltration. In plants without GFP fluorescence after infiltration with the GFP-sensor, siRNA specific to GFP and the target virus sequence could not be detected. In contrast, infiltrated leaves of wild type and transgenic plants showing GFP fluorescence after infiltration revealed accumulation of siRNA specific to the sequence of the sensor. Silencing could be inhibited by co-infiltration using a p19 silencing suppressor construct together with the GFP-sensor, which always resulted in bright GFP fluorescence. In parallel, virus resistance of transgenic Nicotiana benthamiana was investigated via challenge inoculation with GFLV. Our results indicate that efficient RNAi in transgenic plants does not necessarily lead to a detectable accumulation of siRNA.