Abstract
BackgroundViroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence. At present, those dimers are generated by self-ligation of monomeric cDNA, a strategy that presents several disadvantages: (i) low efficiency, (ii) it is a non-oriented reaction requiring tedious screenings and (iii) additional steps are required for cloning into a binary vector for agroinfiltration or for in vitro RNA production.ResultsWe have developed a novel strategy for simultaneous construction of a viroid dimeric cDNA and cloning into a multipurpose binary vector ready for agroinfiltration or in vitro transcription. The assembly is based on IIs restriction enzymes and positive selection and supposes a universal procedure for obtaining infectious clones of a viroid independently of its sequence, with a high efficiency. Thus, infectious clones of one viroid of each family were obtained and its infectivity was analyzed by molecular hybridization.ConclusionThis is a zero-background strategy for direct cloning into a binary vector, optimized for the generation of infectious viroids. As a result, this methodology constitutes a powerful tool for viroid research and exemplifies the applicability of type IIs restriction enzymes and the lethal gene ccdB to design efficient and affordable direct cloning approaches of PCR products into binary vectors.
Highlights
Viroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence
Dimeric clones construction In order to enable the direct cloning of viroid dimeric cDNAs, a suitable vector for generating transcripts was designed
The process consists of direct cloning into a binary vector that can be transformed into Agrobacterium tumefaciens to establish viroid infection by agroinfiltration or employed to generate RNA transcripts in vitro using T7 RNA polymerase
Summary
Viroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence. Viroids are small single-stranded plant-pathogenic RNAs, being considered the smallest (246–401 nt) autonomous infectious nucleic acids known so far [1]. More than 50 species of viroids have been described, being currently grouped into the families Pospiviroidae and Avsunviroidae, based on their replication site (nuclei and chloroplasts, respectively), presence of particular sequence domains, and properties of their infectious cycle [4]. In both groups, replication takes place through a rolling-circle mechanism. The study of viroids might still help to elucidate key biological pathways in plants such as RNA traffic [9,10,11] or genetic regulation by epigenetic modifications [12,13,14]
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