BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells

Abstract

Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs alpha, beta and gamma 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 beta, encoding a non-structural protein, was replaced by the firefly luciferase (luc) reporter gene to yield RNA beta 2-luc. In the presence of both RNAs alpha and gamma, RNA beta 2-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 beta and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern analysis, indicating that the high level of luc gene expression using RNA beta 2-luc resulted from RNA amplification. ORFa of RNA beta, encoding the coat protein, was also replaced by the luc gene to yield RNA beta 1-luc. Although transfection of RNA beta 1-luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further increase of luciferase activity was observed in the presence of RNAs alpha and gamma, or RNAs alpha, beta and gamma, suggesting that the deleted sequences within ORFa are cis-acting for replication of RNA beta. 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.