title: Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco.
abstract: External application of dsRNA molecules from Tobacco mosaic virus (TMV) p126 and CP genes confers significant resistance against TMV infection. Exogenously applied dsRNA exhibits a rapid systemic trafficking in planta , and it is processed successfully by DICER-like proteins producing small interfering RNAs. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA), which protects eukaryotic cells against invasive nucleic acids like viruses and transposons. In the present study, we used a non-transgenic strategy to induce RNAi in Nicotiana tabacum cv. Xanthi plants against TMV. DsRNA molecules for the p126 (TMV silencing suppressor) and coat protein (CP) genes were produced by a two-step PCR approach followed by in vitro transcription. The application of TMV p126 dsRNA onto tobacco plants induced greater resistance against TMV infection as compared to CP dsRNA (65 vs. 50 %). This study also reported the fast systemic spread of TMV p126 dsRNA from the treated (local) to non-treated (systemic) leaves beginning from 1 h post-application, confirmed by both conventional and real-time RT-PCR. Furthermore, we employed a stem-loop RT-PCR and confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves post-application. The approach employed could represent a simple and environmentally safe way for the control of plant viruses in future agriculture.
authors.affiliation: Laboratory of Plant Breeding and Biometry, Agricultural University of Athens, 11855, Athens, Greece.|Department of Virology, Sri Venkateswara University, Tirupati, Andhra Pradesh, 517502, India.
authors.forename: Naga Charan
authors.initials: NC
authors.lastname: Konakalla
authors.affiliation: Laboratory of Plant Breeding and Biometry, Agricultural University of Athens, 11855, Athens, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Kaldis
authors.affiliation: Laboratory of Plant Breeding and Biometry, Agricultural University of Athens, 11855, Athens, Greece.
authors.forename: Margarita
authors.initials: M
authors.lastname: Berbati
authors.affiliation: Department of Virology, Sri Venkateswara University, Tirupati, Andhra Pradesh, 517502, India. hemamasarapu70@gmail.com.
authors.forename: Hema
authors.initials: H
authors.lastname: Masarapu
authors.affiliation: Laboratory of Plant Breeding and Biometry, Agricultural University of Athens, 11855, Athens, Greece. avoloud@aua.gr.
authors.forename: Andreas E
authors.initials: AE
authors.lastname: Voloudakis
mesh_terms: D036022:Capsid Proteins / Q000235:genetics*; D060467:Disease Resistance / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D054884:Host-Pathogen Interactions / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D018515:Plant Leaves / Q000235:genetics / Q000821:virology; D034622:RNA Interference; D034443:RNA Transport; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014026:Nicotiana / Q000235:genetics* / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics*
chemical_list: C071899:130k protein, Tobacco mosaic virus; D036022:Capsid Proteins; C009496:H protein, Tobacco mosaic virus; D012330:RNA, Double-Stranded; D014764:Viral Proteins
keywords: Coat protein; Double-stranded RNA; RNA interference; Silencing suppressor; Systemic transport; Tobacco mosaic virus; p126; siRNAs