Heterologous expression of circular RNAs in Escherichia coli for analyzing the ligation process of chloroplastic viroids and producing double-stranded RNAs with insecticidal activity

Abstract

Viroids, with a minimal genome of highly structured, single-stranded, circular noncoding RNA, can parasitize plant cell components to replicate autonomously, establish systemic infections and usually cause diseases. Those of the family Avsunviroidae replicate and accumulate in chloroplasts through a symmetric rolling circle mechanism, in which a chloroplast RNA polymerase produces linear concatemers of complementary polarity that are reduced to monomeric intermediates by the activity of hammerhead ribozymes (HHR) present in the concatemer. The 5'-hydroxyl and 2',3'-cyclic phosphodiester ends generated are substrates in the formation of an intramolecular 5',3'-phosphodiester bond catalyzed by the chloroplastic isoform of tRNA ligase, generating circular molecules of complementary polarity that can enter another round of transcription, symmetrical to the first. In this Thesis the viroid sequences and structures that are essential for circularization have been analyzed, using as a model the eggplant latent viroid (ELVd), which induces asymptomatic infections in eggplant (Solanum melongena L.). To do this, we expressed in Escherichia coli linear ELVd (+) precursors flanked by two copies of their HHR. Its processing produces monomers with