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Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus

Ibrahim, AEC; Van Dolleweerd, CJ; Drake, P; Ma, J (2020) Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus. PLoS One, 15 (3). e0229877. ISSN 1932-6203 https://doi.org/10.1371/journal.pone.0229877
SGUL Authors: Drake, Pascal Ma, Julian

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Abstract

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Item Type: Article
Additional Information: Copyright: © 2020 Ibrahim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: MD Multidisciplinary, General Science & Technology
SGUL Research Institute / Research Centre: Academic Structure > Infection and Immunity Research Institute (INII)
Journal or Publication Title: PLoS One
ISSN: 1932-6203
Dates:
DateEvent
5 March 2020Published
15 February 2020Accepted
Publisher License: Creative Commons: Attribution 4.0
Projects:
Project IDFunderFunder ID
WT093092MAWellcome TrustUNSPECIFIED
ERC-2010-ADG_20100317European Research CouncilUNSPECIFIED
UNSPECIFIEDThe Hotung FoundationUNSPECIFIED
URI: http://openaccess.sgul.ac.uk/id/eprint/111697
Publisher's version: https://doi.org/10.1371/journal.pone.0229877

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