Episomal expression of a hammerhead ribozyme directed against plum pox virus

Two related antisense RNAs directed against plum pox virus (PPV) were expressed episomally in Nicotiana clevelandii by infection with recombinant potato virus X (PVX). One recombinant PVX expressed an ordinary PPV antisense RNA of about 400 nucleotides, while the other expressed a related antisense RNA that carried the catalytic domain of a hammerhead ribozyme. Inoculation with the latter recombinant PVX resulted in the accumulation of ribozyme RNA that was catalytically active when tested in vitro with a PPV substrate RNA. Plants that had been inoculated with recombinant PVX viruses, expressing either PPV-directed antisense or ribozyme sequences or GUS RNA as a control, were challenged with PPV by a sequential second inoculation. In plants that expressed PPV antisense sequences, the appearance of PPV disease symptoms was delayed for 3-5 days. Quantification of PPV 1 week after inoculation showed that the protective effect by the episomally expressed catalytic antisense RNA was stronger than that of the ordinary antisense RNA. However, eventually all plants tested accumulated comparable titers of PPV.