Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections

Francisco Tenllado,José Díaz-Ruíz,Belén Martínez-García,Marisol Vargas

doi:10.1186/1472-6750-3-3

Francisco Tenllado, José Díaz-Ruíz + Show 2 more

Open Access

https://doi.org/10.1186/1472-6750-3-3

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Abstract

BackgroundDouble-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.ResultsWe have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation.ConclusionOur approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

Highlights

Double-stranded RNA is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals
Production of virus-derived Double-stranded RNA (dsRNA) using an RNase-deficient E. coli strain E. coli strain HT115(DE3) was chosen to take advantage of an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase gene contained within a stable insertion of a modified lambda prophage λ DE3 [22]
Upon induction with 0.4 mM IPTG, dsRNA produced in these bacteria was analyzed by preparation of total nucleic acid followed by treatment with RNase A to remove single-stranded RNA (Fig. 1)

Summary

Introduction

Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. Posttranscriptional gene silencing (PTGS) in plants is a homology-dependent RNA degradation system designed to act as a natural defence barrier against virus infections [1]. Induction of RNA silencing in different organisms can be activated by exogenously supplied double-stranded RNA (dsRNA) [10,11]. In this sense, RNAi has become a powerful genetic tool for selectively silencing gene (page number not for citation purposes)

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