Infectious Barley stripe mosaic virus RNA transcribed in Vitro from full-length genomic cDNA clones

Abstract

Full-length genomic cDNA clones of the Type and ND18 strains of barley stripe mosaic virus (BSMV) were transcribed in vitro using T7 RNA polymerase. The combination of RNAs α, β, and γ synthesized in the presence of 5′ cap analogs was infectious after inoculation onto barley plants, conclusively demonstrating the tripartite nature of the BSMV genome. Transcripts synthesized in the absence of cap analogs were not infectious. A γ-specific subgenomic RNA which is normally present in BSMV virions was not required to establish a systemic infection. In vitro transcripts of variant cDNA clones which were isolated from the ND18 strain, containing either a simple nucleotide substitution or a 372-nucleotide duplication similar to one found in the genome of the Type strain, were also found to be biologically active. Two dicotyledonous hosts which have a differential response to infection with the Type and ND18 strains of BSMV were identified and these phenotypes were shown to be faithfully reproduced by inoculation with in vitro transcripts derived from the appropriate full-length cDNA clones.