Quantifying Plum Translation Factor Expression

Total RNA was extracted from leaves of PPV-challenged transgenic lines collected in the third cycle of PPV testing. They were treated with Turbo DNaseI (Thermo Fisher Scientific) before complementary DNA synthesis with the Superscript II® reverse transcriptase from Invitrogen/ Revertaid/Ribolock reverse transcriptase kit (Fermentas), using an oligo-dT (16) primer. Relative qPCR to quantify PpeIF4E, PpeIF4G, PpeIFiso4G10 and PpeIFiso4G11 mRNA accumulation was performed on a Light Cycler 480 II machine (Roche Diagnostics) by using LightCycler® 480 SYBR Green I master and one tenth of the newly synthesized cDNAs. Expression of TEFII (Prupe.4G138700) was used as internal reference. Based on re-sequenced Japanese plum copies, specific primers were designed for each copy of the Prunus translation initiation factor genes as well as for the internal reference gene (Additional file 10: Table S4). RT-qPCR procedures for cycling conditions and relative expression statistical analysis are detailed elsewhere [53 (link)]. Values statistically different when comparing the expression level of wild-type and transgenic lines were verified by analysis of summary rank with the Kruskal-Wallis test with the R v 3.2.5 software.