Silencing on the Spot. Induction and Suppression of RNA Silencing in the Agrobacterium-Mediated Transient Expression System

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Abstract
The Agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants. In many cases, high levels of active protein can be produced without the need to produce transgenic plants. In this study, a series of tools were developed to enable strong or weak induction of RNA silencing and to suppress RNA silencing in the absence of stable transgenes. Transient delivery of a gene directing production of a double-stranded green fluorescent protein (GFP) transcript rapidly induced RNA silencing of a codelivered GFP reporter gene, effectively preventing accumulation of GFP protein and mRNA. RNA silencing triggered by the strong dsGFP inducer was partially inhibited by the tobacco etch virus silencing suppressor, P1/HC-Pro. In the absence of the strong double-stranded GFP inducer, the functional GFP gene served as a weak RNA silencing inducer in the transient assay, severely limiting accumulation of the GFP mRNA over time. The weak silencing induced by the GFP gene was suppressed by P1/HC-Pro. These results indicate RNA silencing can be triggered by a variety of inducers and analyzed entirely using transient gene delivery systems. They also indicate that RNA silencing may be a significant limitation to expression of genes in the Agrobacterium-mediated transient assay but that this limitation can be overcome by using RNA silencing suppressors.