Generation of efficient resistance to Plum pox virus (PPV) in Nicotiana benthamiana and Prunus domestica expressing triple-intron-spanned double-hairpin RNAs simultaneously targeting 5’ and 3’ conserved genomic regions of PPV.

Plum pox virus (PPV) is the causal agent of the devastating viral disease, known as sharka in Europe, on many stone fruit species. To engineer genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach, previously two plant transformation vectors, pAWp1 and pAWcp were constructed by cloning two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, into a Ti binary vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo-polygalacturonase (endo-PG) genomic DNA. The resulting transgenic plants expressing hairpin RNAs either targeting the viral P1 or CP sequences showed resistance to PPV. Here we report construction of a new construct pAWp1-cp by cloning the P1 and CP hairpin sequences together into the Ti vector and the inserted DNA contains a triple-intron double-hairpin sequence simultaneously targeting the P1 and CP sequence of PPV. This vector was transformed into Nicotiana benthamiana and plum (Prunus domestica L.). Resistance assays showed the transgenic plants were efficiently resistant to PPV.