Towards developing resistance to Citrus tristeza virus (CTV) by expressing using pokeweed antiviral protein in transgenic grapefruit

Pokeweed antiviral protein (PAP) isolated from Phytolacca Americana has been shown to have a broad range antiviral activity. For in vivo experiments, the wild type PAP gene was cloned into a plant transformation vector. We also prepared a DNA construct which produced a deletion of 25 amino acids from the C-terminus of PAP, and two constructs with independent point mutations at the N-terminus (Gly75-Asp), and the active site (Glu176-Ala). These four constructs were then transformed into Citrus paradisi by Agrobacterium-mediated transfer. Nine transgenic plants containing the N-terminal mutant, one containing the active site mutation, and none with the other two constructs were obtained and characterized by molecular methods. The transgenic citrus plants are being graft- and aphid-challenged with CTV and will be analyzed for CTV replication.