|Choi, I. - UNIVERSITY OF NEBRASKA|
Submitted to: International Congress of Virology
Publication Type: Abstract Only
Publication Acceptance Date: April 10, 1999
Publication Date: August 6, 1999
Citation: French, R.C., Choi, I.R., Stenger, D.C. 1999. A stable cdna clone of wheat streak mosaic virus yields infectious in vitro transcripts. International Congress Of Virology. (Meeting-not published.) Technical Abstract: Wheat streak mosaic virus (WSMV) is an important viral pathogen of wheat in the Great Plains region of North America and also has been reported to occur in Europe and the Middle East. WSMV is transmitted by eriophyid mites and has the same genome organization as aphid-transmitted potyviruses. A full-length cDNA clone of WSMV was constructed in pUC18 with an upstream SP6 promoter and the 3'-end was followed by an unique SacII site for linearizing the plasmid. In vitro transcripts were generated and used to inoculate wheat seedlings. More than 60% of the plants inoculated with transcripts produced in the presence of 7-methyl-guanosine cap analog developed mosaic symptoms typical of WSMV infection. The presence of virus in systemically infected leaves of inoculated plants was confirmed by RT- PCR of the WSMV P3 gene, and by serological detection of WSMV coat protein on immunoblots. Uncapped transcripts were not infectious. The pUC18 construct containing WSMV cDNA was unstable and prone to spontaneous rearrangement during culture in E. coli, particularly upon subculturing. A parallel cDNA clone was constructed in the low copy number plasmid pACYC177 which proved to be much more stable during culture in E. coli. Capped transcripts from the pACYC177 based cDNA clone were also infectious, leading to systemic infection of about 50% of inoculated wheat plants. This is the first report of biologically active in vitro transcripts from cDNA of an eriophyid mite-transmitted member of the family Potyviridae and should be a valuable tool to establish WSMV gene expression and function.