Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus

Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DN A that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plant s inoculated with in vitro transcripts was confirmed by reverse-transcripti on polymerase chain reaction of the WSMV P3 gene and by accumulation of WSM V coat protein as detected by immunoblotting. Maintenance of the full-lengt h WSMV cDNA in the high copy number plasmid pUC18 was problematic because o f spontaneous rearrangement of WSMV sequences during growth in liquid media for more than similar to 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to I liter) of liquid culture for similar to 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mit e vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyled onous hosts for which infectious in vitro transcripts are available.