Abstract
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.
Highlights
RNA-based gene silencing is a sequence-specific, conserved mechanism in eukaryotes implicated in viral defense, control of transposable elements, and gene regulation
To investigate the impact of the partial transposase sequence on systemic silencing in the 16C line, we synthesized T-DNA inserts containing either the full T-DNA sequence reported for the 16C line [31] or the same T-DNA sequence without the partial transposon
The T-DNAs were cloned into binary vectors and the cassette sequence was confirmed. pMON417669 comprised the insert including the selectable marker, the Green Fluorescent Protein (GFP) expression cassette, and the partial transposase. pMON417670 comprised the same sequence without the partial transposase sequence (Fig 1A)
Summary
RNA-based gene silencing is a sequence-specific, conserved mechanism in eukaryotes implicated in viral defense, control of transposable elements, and gene regulation. Gene silencing using transgenic approaches have been utilized to deploy a number of agriculturally important traits including virus resistance in papaya [1], delayed fruit ripening in tomato [2], black-spot bruise resistance and lower acrylamide levels post-cooking in potato [3], improved oil composition in soybeans [4] and insect control in corn [5]. These commercial products and others like them all take advantage of DCL-like proteins that cleave various forms of dsRNA into small interfering RNAs (siRNAs) 21-24nt in length. This study is part of an effort to understand how topically-delivered 22nt dsRNAs targeting GFP leads to a systemic silencing response in N. benthamiana
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