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.
Highlights
Sir Alexander Fleming Building, Department of Biological Sciences, Imperial College, London SW7 2AZ, Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Centro de Investigacion en Quimica Aplicada, CIQA-CONACYT, Saltillo 25294, Mexico
Overlapping PCR was used to produce full-length cDNAs, which were fragments were amplified from total RNA extracted from Cucurbit yellow stunting disorder virus (CYSDV)-infected plants using Pfu polymerase cloned into pGEM-T-Easy vector (Promega)
LIYV, where where transfection of virion into tobacco protoplasts resulted in h delays in the initiation transfection of virion RNA into tobacco protoplasts resulted in 24 h delays in the initiation and and attainment of maximal replication of RNA2, relative to RNA1 [7]
Summary
Agrobacterium-mediated of plants with LIYV cDNAs promoters, were used to transfect tobacco protoplasts transformation [4]. These clones, in conjunction with later under the control of the. LIYV biology versions permitting Agrobacterium-mediated transformation of plants with LIYV cDNAs under [6]. It was shown that RNA1 is competent to replicate aloneinsights [4], and to LIYV drivebiology the trans-replication of control of the CaMV. RNA2isin an asynchronous manner p34 was shown to be essential that RNA1 competent to replicate alone[7]. RNAs further aspects of crinivirus biology, including the synchronous replication of LCVofRNA1 and RNA2 arising fromand thethe former.
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