Engineering resistance to Plum pox virus (PPV) through the expression of PPV-specific hairpin RNAs in transgenic plants

Plum pox virus (PPV) causes serious diseases in stone-fruit trees, PPV is widespread in Europe and was recently detected in peach orchards in Canada and the United States. In an effort to engineer resistance to PPV in Prunus species, we produced transgenic plants expressing PPV-specific hairpin RNAs under the control of a constitutive promoter. Two highly conserved regions of the PPV genome were introduced into plants as inverted repeats separated by an intron sequence. In initial resistance assays, 22 Nicotiana benthamiana transgenic lines were inoculated with a Canadian isolate of PPV and tested for the presence of PPV between 4 and 5 weeks post inoculation, using the enzyme-linked immunosorbent assay (ELISA) diagnostic method. The majority of the transgenic lines displayed increased resistance to PPV, with only one transgenic line being completely susceptible to the virus. In the second round of resistance assays, a more sensitive detection method, reverse transcription - polymerase chain reaction, was used to test for the presence of PPV at various times post inoculation. The virus was readily detected in inoculated control plants, but not in six selected transgenic lines, suggesting that the latter are highly resistant to PPV. PPV-specific small interfering RNAs were observed in the six selected transgenic lines, confirming that posttranscriptional gene silencing is the mechanism of resistance in these lines. Our results also indicate that the expression of PPV-specific hairpin RNAs is an effective method of inducing resistance to the virus.