Figure 2

Schematic representation of constructing pBD-TuMV infectious clones. (A) Two TuMV in vivo infectious clones, p35S-TuMV-YC5 and p35S-TuMV-GFP (Lin et al., 2009; Niu et al., 2006), constructed on the pCaMVCN vector (Pharmacia) were designed for mechanical inoculation on C. quinoa plants. The two clones were used in the current study to construct a series of infectious constructs using a binary vector for agro-infiltration. (B) The full-length cDNA constructs of TuMV were constructed in the pBD003 binary vector (pBD-TuMV-YC5 and pBD-TuMV-GFP) for agrobacterium infiltration on N. benthamiana plants. The construction steps for cloning the TuMV full-length cDNA into the pBD003 binary vector are shown. The full-length TuMV cDNAs released from the p35S-TuMV series were constructed downstream of the CaMV 35S promoter (35S) by insertions in between suitable restriction enzyme sites of pBD003. The final constructs were designated pBD-TuMV-YC5 and pBD-TuMV-GFP, contained a complete cDNA copy of wild type TuMV and TuMV carrying GFP, respectively. The coding regions of the cDNA are indicated by an open box. The 5′ and 3′ non-coding regions of the cDNA are indicated by heavy lines. (C) R₁₈₂K mutation on HC-Pro of the TuMV was conducted by PCR-mediated mutagenesis. The mutated fragment was digested with MfeI/Kpn I and replaced the corresponding region of pBD-TuMV-GFP to generate pBD-TuMV-GK. An asterisk indicated the R₁₈₂K mutation position.