In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection

Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5′ RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.