RNA Interference Provides High-level, Stable Resistance to Plum Pox Virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to PPV infection through RNA silencing  involving both small interfering RNA  (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host.  Plums inoculated with the major PPV strains, 3 widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy.  PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that can infect cherry species but was unable to infect P. domestica.  This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation. Regardless of PPV strain variability based on geography, host species, epidemiology, serotypes of the CP protein, and substitutions of nucleotides at the NH2-terminus of CP of the PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.