Fig. 1

Schematic representation of viroid dimerization and subsequent assembly in a binary vector. a Two different pairs of primers are used to generate distal BsaI recognition sites (magenta) in such a way that compatible ends for assembly can be obtained. b In a single reaction of simultaneous restriction and ligation the two viroid monomers (blue) are ligated between them and to a binary vector with compatible cohesive ends (orange). c Specifically, the viroid dimer is inserted into an expression cassette that contains and a duplicated 35S, a constitutive promoter for plant expression and a PoPit terminator. Additionally, this binary vector has a T7 promoter for in vitro transcription. The dimeric viroid cDNA sequence replaces a lethal gene ccdB, thus guaranteeing an optimal efficiency of the reaction. d Detail of the generation of the receptor–vector starting from pMD201t construct)