Figure 3

Viral replicon used for in planta studies. Viral replicon was constructed with the pCAMBIA1391Z binary vector. Complete replicon contains the region spanning from CR to AC3 (pCK2 replicon) and CR region of ToLCKeV. Presence of CR on either end in the same orientation enables the completion of rolling circle replication. Rolling circle replication releases the episome that contains only one complete CR and region spanning from AC1 to AC3. Red arrows indicate the nicking site of Rep protein in hairpin loop in either CRs and the black line represents the region of the vector that forms episome. Formation of an episome can be checked by PCR amplification with the oligonucleotides indicated by blue arrows. Internal primers were designed to amplify the DNA only from the episome under standardized PCR conditions. CR-AC3 is replaced by CR-AC3^M or CR-AC3^M21 for generating pCK2^M replicon and pCK2^M21 replicons. A 300 bp PCNA fragment was cloned into the MCS region to generate pCK2^M-PCNA and pCK2^M21-PCNA.