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

• Published in: Plant physiology (Bethesda) Vol. 126; no. 3; pp. 930 - 938
• Main Authors: Johansen, Lisa K., Carrington, James C.
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 01.07.2001; American Society of Plant Physiologists
• Subjects: Agrobacterium; Biological and medical sciences; Biotechnology; Breakthrough Technologies; Cloning, Molecular; Cysteine Endopeptidases - genetics; Double stranded RNA; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Plant; Gene Silencing; Genes; Genetic engineering; Genetic technics; Genetic vectors; Green Fluorescent Proteins; Luminescent Proteins - genetics; Messenger RNA; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular genetics; Nicotiana; Plant Leaves - metabolism; Plants, Toxic; Potyvirus - genetics; Rhizobium - genetics; RNA; RNA interference; RNA, Plant - genetics; Solanaceae - genetics; Solanaceae - metabolism; Tobacco etch virus; Transgenes; Transgenic animals and transgenic plants; Transgenic plants; Viral Nonstructural Proteins - genetics; Viral Proteins - genetics; Viruses; Transcription promoter; Gene expression; Transients; Gene silencing; Regulation(control); Agrobacterium; Investigation method; Dicotyledones; Angiospermae; Bacteria; Suppressor; Spermatophyta; Transgenic plant; Rhizobiaceae; Solanaceae; Technique; Transcription factor; Experimental plant; Gram negative bacteria
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.126.3.930

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.