RESUMEN
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.
Asunto(s)
Crinivirus/genética , Cucumis sativus/virología , ADN Complementario , Protoplastos/virología , ARN Viral/biosíntesis , Replicación Viral , Transcripción Genética , TransfecciónRESUMEN
Potato yellow vein virus (PYVV) is a whitefly-transmitted (Trialeurodes vaporariorum) closterovirus (WTC) with an as yet unidentified genome composition. PYVV dsRNA preparations consist of three high molecular weight dsRNA species (dsRNAs 1, 2 and 3) 8.0, 5.5 and 4.0 kbp in size respectively, as well as two low molecular weight dsRNA species of 2.0 and 1.8 kbp (denoted x and y). The PYVV capsid protein minor (CPm) gene was identified on the dsRNA 3 species, and was subsequently cloned and sequenced. The PYVV CPm gene is 2022 nucleotides long and putatively encodes a protein with estimated size 77.5 kDa. The PYVV CPm gene product is considerably larger than the equivalent proteins encoded by the bipartite criniviruses, Lettuce infectious yellows virus (LIYV) and Cucurbit yellow stunting disorder virus (CYSDV) (52 and 53 kDa, respectively). The PYVV CPm possesses a centralized domain which is absent from both the LIYV and CYSDV CPm counterparts. Pairwise comparisons as well as phylogenetic analysis based on the available amino acid sequences of the CPm of various WTCs, showed that PYVV is closely related to LIYV, CYSDV and also Beet pseudo-yellows virus.