Journal: Plant Methods

Article Title: Highly efficient construction of infectious viroid-derived clones

doi: 10.1186/s13007-019-0470-4

Figure Lengend Snippet: Schematic representation of viroid dimerization and subsequent assembly in a binary vector. a Two different pairs of primers are used to generate distal Bsa I 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)

Article Snippet: Viroid transcripts were generated by transcription of 400 ng of linearized pMD201t HSVd/ELVd (Digested with Eco RI) with T7 RNA polymerase (Takara, Kusatsu, Japan) for 3 h according to manufacturer instructions.

Techniques: Plasmid Preparation, Ligation, Expressing, In Vitro, Sequencing, Construct

Journal: Plant Methods

Article Title: Highly efficient construction of infectious viroid-derived clones

doi: 10.1186/s13007-019-0470-4

Figure Lengend Snippet: Workflow proposed to obtain infectious clones of a viroid. Viroid sequence can be amplified from infected tissue by RT-PCR or from a DNA source by PCR. If the viroid sequence does not contain a bsa I recognition site, the viroidal cDNA can be directly assembled into the binary vector (pMD201t), replacing a lethal gene, in a simultaneous bsa I restriction and ligation. Conversely, if there the viroid contains a bsa I recognition site, it can be cloned using another IIs enzyme, such as Bsm BI. Once digested and purified, the viroid cDNA is dimerized by ligation to a previously digested pMD201t. The dimeric viroidal cDNA cloned into pMD201t (pMD201t-viroid) can be used to generate the infectious RNA transcript in vitro, using T7 RNA polymerase onto a linearized plasmid (digested with Eco RI). Additionally, the pMD201t-viroid can be transformed into Agrobacterium tumefaciens for transient plant transformation and subsequent production of infective RNA in vivo

Article Snippet: Viroid transcripts were generated by transcription of 400 ng of linearized pMD201t HSVd/ELVd (Digested with Eco RI) with T7 RNA polymerase (Takara, Kusatsu, Japan) for 3 h according to manufacturer instructions.

Techniques: Clone Assay, Sequencing, Amplification, Infection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Ligation, Purification, In Vitro, Transformation Assay, In Vivo