Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine
Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.