BSMV GENOME MEDIATED EXPRESSION OF A FOREIGN GENE IN DICOT AND MONOCOT PLANT-CELLS

Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs α, β and γ that have been cloned into in vitro transcription vectors from which infectious transcripts can be obtained. The BSMV genome has been engineered here to serve as an expression vector in plant protoplasts. Open reading frame (ORF) b of RNA β, encoding a non-structural protein, was replaced by the firefly luciferase (luc) reporter gene to yield RNA β2-luc. In the presence of both RNAs α and γ, RNA β2-luc mediated efficient expression of the luc gene upon transfection into tobacco and maize protoplasts. This expression ranged from 20- to 123-fold higher than the luciferase activity obtained from transfection with a luc gene mRNA. Replication of RNA β and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern analysis, indicating that the high level of luc gene expression using RNA β2-luc resulted from RNA amplification. ORFa of RNA β, encoding the coat protein, was also replaced by the luc gene to yield RNA β1-luc. Although transfection of RNA β1-luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further increase of luciferase activity was observed in the presence of RNAs α and γ, or RNAs α, β and γ, suggesting that the deleted sequences within ORFa are cis-acting for replication of RNA β. Our results demonstrate that a foreign gene can be expressed by replacement of a viral non-structural protein gene that is essential for virus multiplication in plants, leading to a potential strategy for virus 'containment' with use of 'disarmed' plant viral vectors.