In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts.
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.
Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepio, Chania GR-73100, Greece. owen@maich.gr.
Carolyn A
CA
Owen
Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepio, Chania GR-73100, Greece. moukarzel.romy@gmail.com.
Romy
R
Moukarzel
Sir Alexander Fleming Building, Department of Biological Sciences, Imperial College, London SW7 2AZ, UK. huang.xiao@gmail.com.
Xiao
X
Huang
Departamento de Biolog&#237;a del Estr&#233;s y Patolog&#237;a Vegetal, Centro de Edafolog&#237;a y Biolog&#237;a Aplicada del Segura (CEBAS)-CSIC, P.O. Box 164, 30100 Espinardo, Murcia, Spain. mona_kassem@yahoo.com.
Mona A
MA
Kassem
Sir Alexander Fleming Building, Department of Biological Sciences, Imperial College, London SW7 2AZ, UK. eeliasco@hotmail.com.
Eleonora
E
Eliasco
Departamento de Biolog&#237;a del Estr&#233;s y Patolog&#237;a Vegetal, Centro de Edafolog&#237;a y Biolog&#237;a Aplicada del Segura (CEBAS)-CSIC, P.O. Box 164, 30100 Espinardo, Murcia, Spain. m.aranda@cebas.csic.es.
Miguel A
MA
Aranda
Department of Biological and Environmental Sciences, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB, UK. r.coutts@herts.ac.uk.
Robert H A
RH
Coutts
Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepio, Chania GR-73100, Greece. livieratos@maich.gr.
Ioannis C
IC
Livieratos
D029383:Crinivirus / Q000235:genetics*; D018553:Cucumis sativus / Q000821:virology*; D018076:DNA, Complementary; D011523:Protoplasts / Q000821:virology*; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
D018076:DNA, Complementary; D012367:RNA, Viral
Cucurbit yellow stunting disorder virus; criniviruses; infectious clones; protoplasts; whitefly-transmitted viruses