Summary
   Viroids, the simplest infectious pathogens, are formed by a single-stranded circular RNA. Despite their small size (246-401 nt) and their inability for coding proteins, they replicate autonomously, move systemically, and incite diseases in certain plants. In the present work we have examined two methods for viroid control based on hammerhead ribozymes with tertiary stabilizing motifs, and on interfering RNAs that induce a plant defensive response.
   Eggplant latent viroid (ELVd) hammerheads, which in its natural context mediate self-cleavage of the multimeric RNA intermediates generated in a rolling-circle mechanism of replication, appear particularly suited for a trans-cleaving design. We have observed that the trinucleotide preceding the self-cleavage site of ELVd (+) hammerhead affects its activity. The catalytic constants of some trinucleotide variants (AUA, AUC, GUA, GUC) measured in vitro at low Mg2+ concentration are different in co- and post-transcriptional conditions. These results suggest that the ELVd (+) hammerhead (and most likely other natural hammerheads) have been selected during evolution for acting during transcription, and that the AUC trinucleotide preceding the self-cleavage site is excluded from the majority of natural hammerheads for adopting catalytically-inactive metastable structures during transcription. The ELVd(+)-GUC variant, which has a high catalytic constant and induces cleavage of a large fraction of the primary transcript, was chosen for designing trans-cleaving variants against Potato spindle tuber viroid (PSTVd) RNA.
   Trans-cleaving hammerheads with discontinuous or extended format and tertiary stabilizing motifs derived from ELVd (+) and Peach latent mosaic viroid (PLMVd) (-) hammerheads catalyzed cleavage of a structured PSTVd RNA of 464 nt in vitro at physiological Mg2+ concentration. The most efficient ribozyme had an extended format and was derived from the PLMVd (-) wild-type hammerhead. Agroinfiltration experiments in N. benthamiana in which recombinant plasmids expressing this later hammerhead and a PSTVd RNA substrate, showed a decrease of substrate accumulation in the infiltrated leaves with respect to a control with a similar hammerhead lacking the tertiary stabilizing motifs. When the stabilized hammerhead was expressed simultaneously with an infectious PSTVd RNA the accumulation of the genomic viroid RNA in the upper non-infiltrated leaves was delayed.
   Finally, mechanical co-inoculation of viroid genomic RNAs with an excess of their homologous dsRNAs led to a significant reduction of infectivity in four different systems [Citrus exocortis viroid (CEVd)-gynura, CEVd-tomato, PSTVd-tomato and Chrysanthemum chlorotic mottle viroid (CChMVd)-chrysanthemum] which include members of both viroid families. The effects were sequence specific, depended on the plant growing temperature and, in some cases, on the dose of dsRNAs. A similar effect was observed in the CEVd-gynura and CEVd-tomato systems when the viroid was co-inoculated with an excess of homologous viroid-small RNAs generated in vitro. Moreover, co-agroinfiltration of recombinant plasmids expressing an infectious PSTVd RNA and an homologous hairpin RNA (from a construct with an inverted repeat sequence from PSTVd separated by an intron) delayed viroid accumulation in the infiltrated and in the upper non-infiltrated leaves. The specificity of the protection afforded by these two different methodologies (co-inoculation with dsRNAs and transient expression of hairpin RNAs) suggests that, in certain conditions, viroids are substrates of RISC.