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Ribozymes are RNA molecules that catalyze an intramolecular self-cleavage reaction. Hammerheadtype ribozyme has been engineered to work in trans by Haseloff and Gerlach3). The molecule possesses a 22-mer catalytic domain embedded within flanking sequences that are complementary to target RNA sequences. The only sequence required for its cleavage activity in the target RNA sequence is GUX trinucleotides (X=A, U or C), which is common in RNAs. Subsequently, it was shown that the first G can be replaced by any other bases10). Because of its portability and flexibility, hammerhead ribozyme has been successfully used as a regulator of gene expression in mouse6), Drosophila13), tobacco12) and human immunodeficiency virus11). Although plant virus RNAs can also be cleaved by ribozymes in vitro5), few studies have reported in vivo expression of ribozymes for controlling plant virus infection1,2). We report here the construction of hammerhead ribozymes designed to cleave RNA encoding the viral movement protein (3a) of cucumber mosaic virus (CMV) and its activity in vitro and demonstrate the anti-CMV-infection activity of the ribozymes in vivo, by comparing transgenic plants expressing the antisense RNA containing the ribozymes (catalytic antisense RNA) with the plants expressing simple (non-catalytic) antisense RNA or coat protein. We constructed three catalytic antisense fragments, which include one or two catalytic domains (Fig. 1A). Two triplets within 3a gene, GUC at nucleic acid position 159-161 and GUU at 570-572, were chosen for the target sites because they are located within highly conserved region among several strains of CMV. Insertion of catalytic sequence (5•Œ-CTGATGAGTCCGTGAGGACGAA-3•Œ), originating from the genome of the satellite RNA from tobacco ringspot virus, into the antisense cDNA of 3a gene was performed by recombinant polymerase chain reaction (PCR)4) using plasmid pCM3148) carrying full-length cDNA of CMV-Y RNA3 as a template. Amplified catalytic antisense (860bp) fragment was inserted into the BamHI-SacI sites of the RNA expression vector pT7/T3ƒ¿-19(BRL). Resulting plasmid, designated pRz2, pRz3 and pRz4, were linearized with EcoRI and the ribozymes were transcribed by T7 RNA polymerase according to the Stratagene protocol (RNA Transcription Kit) for the purpose of confirming their catalytic activity in vitro. Ten ƒÊg of total RNA prepared from purified CMV virion was mixed with an excess of in vitro transcribed catalytic antisense RNA and incubated in 25ƒÊl of 50mM Tris-HCl (pH 8.0) and 20 mM MgCl2 for 30min at 30•Ž. Reaction products were fractionated on 1.5% agarose gel under denaturing conditions (Fig. 1B). As expected, all ribozymes with heat denaturation prior to the incubation together with the substrate cleaved CMV-RNA3 in the predicted positions. Without heat denaturation, however, reaction rate was significantly reduced (data not shown), indicating that the ribozyme and/or CMV-RNA3 might fold in stable, inactive structures under the in vitro cleavage conditions. To determine whether the ribozyme expressed in plant cells could inhibit viral infection in vivo, we introduced the ribozyme sequence into tobacco plants by Agrobacterium-mediated gene transfer. The ribozyme fragments were excised from pRz2, pRz3 and pRz4, and inserted into the BamHI-SacI sites of binary vector

Resistance to Viral Infection in Transgenic Plants Expressing Ribozymes Designed against Cucumber Mosaic Virus RNA3.