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Viruses 2016, 8(6), 170; doi:10.3390/v8060170

In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

1
Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepio, Chania GR-73100, Greece
2
Sir Alexander Fleming Building, Department of Biological Sciences, Imperial College, London SW7 2AZ, UK
3
Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, P.O. Box 164, 30100 Espinardo, Murcia, Spain
4
Centro de Investigacion en Quimica Aplicada, CIQA-CONACYT, Saltillo 25294, Mexico
5
Department of Biological and Environmental Sciences, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB, UK
*
Author to whom correspondence should be addressed.
Academic Editor: Thomas Hohn
Received: 22 March 2016 / Revised: 24 May 2016 / Accepted: 6 June 2016 / Published: 14 June 2016
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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Abstract

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. View Full-Text
Keywords: criniviruses; whitefly-transmitted viruses; Cucurbit yellow stunting disorder virus; infectious clones; protoplasts criniviruses; whitefly-transmitted viruses; Cucurbit yellow stunting disorder virus; infectious clones; protoplasts
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Owen, C.A.; Moukarzel, R.; Huang, X.; Kassem, M.A.; Eliasco, E.; Aranda, M.A.; Coutts, R.H.A.; Livieratos, I.C. In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts. Viruses 2016, 8, 170.

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