Targeting tobacco mosaic virus with a hairpin ribozyme
Galasinski, Scott C.
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In vitro studies suggest that catalytic RNA may down regulate gene expression through cleavage of target RNA. This application is being evaluated by using a "hairpin" ribozyme derived from the negative strand of the satellite RNA of tobacco ringspot virus. This synthesized ribozyme targets the 542 position of the tobacco mosaic virus (TMV) replicase gene. As a control to verify catalysis as opposed to antisense binding, the ribozyme will be compared to a mutant ribozyme containing a substitution in the hairpin which destroys cleavage activity while retaining the target complement. All constructions are expressed by the CaMV 35S promoter and have been mobilized into the binary vector pBI 121. These vectors have been used in Agrobacterium mediated stable transformations of two Nicotiana tabacum cultivars: Xanthi and Xanthi-nc. These cultivars respond differently to TMV infection and were used to evaluate ribozyme activity by monitoring changes in systemic spread and local lesion response, respectively. Two generations of tobacco expressing the ribozyme were tested for their ability to inhibit TMV. Both the active and mutant ribozymes showed protection when compared to non-transformed plants. There was no statistical difference in viral inhibition between the active or mutant ribozymes in either transformant generation. This suggests that protection is probably occurring through an antisense mechanism.