Expression of single-chain variable fragments against structural and non-structural proteins of Plum pox virus in transgenic Nicotiana benthamiana plants to interfere with viral infection
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Single chain variable fragments (scFv) specific to the coat protein (CP) (scFv4C) and the RNA replicase NIb (scFv2A) from Plum pox virus (PPV) were engineered. Diverse expression versions addressed to different cell compartments were produced by addition of several peptides. Two cytosolic versions of scFv4C with and without KDEL tetrapeptide (scFv4CKDEL) were generated, as well as, three scFv2A versions: one cytosolic (scFv2A), one nuclear (NLS-scFv2A), and a cytosolic facing endoplasmic reticulum membrane targeted version (6K2-scFv2A). Transgenic Nicotiana benthamiana plants expressing these different scFv versions were obtained and some lines from each construct were selected for viral challenge. Challenge experiments were performed by mechanical inoculation with a PPV-D/NAT isolate at moderate inoculum doses (< 0.36 mug/ml viral concentration). The percentage of infected plants was significantly lower in some transgenic lines compared to control plants. Nuclear version of scFv2A and cytosolic version with KDEL of scFv4C showed the highest reduction in viral infection.