Highly efficient construction of infectious viroid-derived clones

Abstract

Background: Viroid 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. Results: We 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. Conclusion: This 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.