|Choi, Il-Ryong - UNIVERSITY OF NEBRASKA|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 26, 1999
Publication Date: December 1, 1999
Citation: Choi, I., French, R.C., Stenger, D.C. 1999. Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus. Phytopathology 89:1182-1185. Interpretive Summary: Wheat streak mosaic virus (WSMV) is an economically important plant virus responsible for economic loss in wheat throughout the Great Plains. The complete genome of WSMV has been assembled in a single DNA clone. RNA produced from the DNA clone was infectious when inoculated to wheat plants and produced both symptoms of wheat streak mosaic disease and progeny virus sparticles that were identical in pathogenic properties to the original WSM isolate used to construct the DNA clone. Progeny virus derived from cloned DNA was transmissible by the natural vector of WSMV, the wheat curl mite. The full-length DNA clone of WSMV was unstable when propagated in certain E. coli plasmids and strains, but could be stablilzed by attention to specific culture growth parameters, E. coli strains, and plasmid vectors used to grow the DNA clone. This represents the first infectious DNA clone of any mite transmissible plant virus, and will facilitate future studies designed to address WSMV gene function and the viral determinants of mite transmission.
Technical Abstract: 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 DNA that had not undergone spontaneous rearrangement during amplification in E. coli, and in vitro transcripts that possessed a 7-methyl-guanosine cap analogue. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by RT-PCR of the WSMV P3 gene, and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic due to spontaneous rearrangement of WSMV sequences upon growth in liquid media for more than ~8 hr, 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 could be grown in large scale volumes (up to 1 L) of liquid culture for ~14 hr without noticeable rearrangements. Both the original WSMV culture and progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite- transmitted plant virus and also represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.