Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana

Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50-72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana.