The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants

Four ribozyme and antisense genes targeting citrus exocortis viroid (CEVd) positive- and negative-strand RNA molecules were constructed and used to transform the tomato Lycopersicon lycopersicum cv. UC82B. The tomato is a readily transformable plant and will support replication of CEVd following mechanical inoculation. The ribozyme genes contained three hammerhead catalytic motifs with long hybridizing arms and synthetic RNA transcripts were shown to cleave the target CEVd RNA molecule in vitro. Homozygous transgenic plants were produced from independent transformants expressing either ribozymes or antisense constructs. Inoculation of transgenic seedlings expressing antisense constructs targeting the negative-strand CEVd RNA molecule with CEVd resulted in a moderate reduction in the accumulation of CEVd RNA. In contrast, similarly inoculated transgenic plants expressing constructs targeting the positive-strand CEVd RNA molecule resulted in an increase in the rate of CEVd RNA accumulation. Addition of the ribozyme motifs to the antisense genes did not enhance their efficiency in the suppression of viroid replication and a moderation or elimination of the observed antisense effects was seen in plants expressing the corresponding catalytic RNA-encoding genes.