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.
Highlights
Lettuce necrotic yellows virus (LNYV) is a plant cytorhabdovirus with a negative-sense, singlestranded, RNA genome that infects lettuce and garlic [1, 2]
We have investigated the feasibility of a reverse genetic system for LNYV based on a negative-sense MG cassette
The advantage of reverse genetic systems based on negative-sense constructs is that it excludes any possibility of MG cassette basal expression independent of the N, P, and L proteins
Summary
Lettuce necrotic yellows virus (LNYV) is a plant cytorhabdovirus with a negative-sense, singlestranded, RNA genome that infects lettuce and garlic [1, 2]. LNYV genomic RNA (gRNA) encodes six monocistronic genes: Nucleoprotein (N), Phosphoprotein (P), cellto-cell movement protein (4b), Matrix protein (M), Glycoprotein (G), and Large Polymerase (L), flanked by the untranslated 3’ leader and 5’ trailer regions [5]. As with other members of the Rhabdoviridae family, gRNA coiled with N protein acts as the template for de novo mRNA synthesis by the viral RNA-dependent RNA polymerase [6]. The viral transcription and replication processes are controlled by cis-acting sequences in the leader (le) and trailer (tr) regions [7,8,9].
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