Figure 3

Reverse-transcription polymerase chain reaction (RT-PCR) was performed to amplify sense and antisense PLRV_MP sequences from the leaves of infected potato plants. (A) Amplified cDNA fragments were analyzed by electrophoresis on 0.8% agarose gel. M: 100 bp DNA ladder; lanes 1 and 2: PCR products of sense and antisense MP (471 bp). (B) The pHANNIBAL plasmids were purified from E. coli DH5a cultures and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lanes 1: pHANNIBAL plasmid (5.83 kb). (C) The pART27 binary plasmid was purified from E. coli DH5a culture and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lane 1: pART27 plasmid (11.6 kb). (D) Confirmation of siRNA constructs in pHANNIBAL and pART27 binary vector through restriction analysis. M: 1 kb DNA ladder; lane 1: undigested pHANNIBAL; lane 2: antisense MP ligated with pHANNIBAL (pHANNIBAL-antisense MP construct); lane 3: both antisense and sense MP ligated with pHANNIBAL (pHANNIBAL-MP-siRNA construct); lane 4: HindIII and BamHI digestion of pHANNIBAL- antisense MP construct shows the release of a 471 bp fragment; lane 5: XhoI and KpnI digestion of pHANNIBAL-MP siRNA construct also shows the release of 471 bp fragments; lane 6: XhoI and BamHI digestion of pHANNIBAL-MP siRNA construct shows the release of a ~ 1500 bp fragment; and lane 7: NotI digestion of pHANNIBAL-MP siRNA construct shows the release of two fragments of ~ 4 kb (including sense MP, intron, and antisense MP sequence) and 3.5 kb. (E) The whole siRNA cassette (~ 4 kb) was transferred from pHANNIBAL to pART27, and this was further confirmed by NotI restriction analysis. (F) Schematic representation of pART27-MP siRNA constructs used for transient expression by agroinfiltration.