Development and evaluation of a GFLV inverted repeat construct for genetic transformation of grapevine

Grapevine Fanleaf virus (GFLV) is one of the most damaging and widespread nepovirus in grapevine, causing the fanleaf disease. Here, we report the development of inverted repeat (IR) constructs, consisting of GFLV-derived sequences, for genetic transformation of grapevine to induce GFLV-specific silencing. The IR construct was designed with fragments of a conserved region of the GFLV movement protein (MPc) gene from a Tunisian isolate (GFLV tun) and with cassettes containing the neomycin phosphotransferase or bar gene as selectable marker. For proof of concept, the IR construct was transferred into Nicotiana benthamiana by Agrobacterium mediated transformation. Challenge inoculation of T1 transgenic plant lines with the relevant virus resulted in plants showing resistance, recovery, retarded infection and susceptibility. Northern blot analysis, using a virus-derived sequence (MPc) as probe, could detect transgene specific small interfering RNA in resistant transgenic N. benthamiana plants after GFLV inoculation. Embryogenic cells of grapevine (Vitis vinifera cv Arich dress,) were transformed with the IR construct in combination with the bar gene using A. tumefaciens strain LBA4404. After molecular characterization of regenerated putative transgenic grapevine plants, Northern blot analysis detected different levels of IR transcripts in independent transgenic grapevine lines indicating a post-transcriptional gene silencing.