RNA extraction, RT-PCR detection and qPCR analysis

Total RNA was prepared using fresh tissue from both leaves and the base of corolla tubes of N. benthamiana flowers, laminar abscission zones of Citrus clementina leaves and Arabidopsis flower receptacles using the TRIZOL method [28]. Quality of the isolated total RNA was checked and quantified using the NanoDrop spectrophotometer (Thermo Fisher Scientific, Alcobendas, Spain).

The systemic infection of N. benthamiana plants was detected at 18 dpi by conventional RT-PCR using primer pairs KU17L/KU7L flanking the insertion site of the CLBV viral vector (Additional file 5). PCR products were visualized by 2% agarose gel electrophoresis and GelRed-staining (Biotium Inc., Hayward, CA, U.S.A.).

Quantitative PCR analysis was performed in three technical replicas using LightCycler® FastStart DNA MasterPLUS SYBR Green I reaction mix and a LightCycler 2.0 instrument (Roche, Basel, Switzerland), using primers for NbenIDA1A and NbenPP2A listed in Additional file 5. The fluorescence intensity data was obtained through LightCycler Software version 4.1. Three biological replicates for corollas from control and clbv3’pr-NbenIDA1 inoculated plants were used. The relative quantification of transcript levels was normalized using the NbenPP2A gene [58] against the control and determined using the 2^−ΔΔCt method.