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Plum pox virus (PPV) is the most devastating viral disease of the stone fruits worldwide. Inefficiency of the traditional control measures against PPV along with its globally widespread distribution and the economic importance of stone fruits, signify the necessity and importance of PPV resistance programs. In the present study, Agrobacterium-mediated transformation of Nicotiana benthamiana Domin was performed using four inverted repeat constructs derived from UTR/P1, HCPro, HCPro/P3, and CP regions of PPV-T isolate KyEsAp301. The efficacy of the constructs for inducing virus resistance in transgenic plants was evaluated by inoculation with PPV-D, -M, and -T strains. The potential of hairpin structures in the production of siRNAs and miRNAs in both wild-type and transgenic plants was compared by small RNA high-throughput sequencing. Although the four PPV genomic regions were used for transgenic resistance in previous experiments, small RNA high-throughput sequencing was first time used in this study to demonstrate the efficacy of the PPV constructs and to determine expression profiles of siRNAs and miRNAs. The results revealed that the potentials of hairpin constructs in producing siRNAs and their accumulation in target regions were significantly different. Expression profiles of several known and novel miRNAs were dramatically changed in response to PPV infection in both wild-type and transgenic plants, demonstrating plausible involvement of these miRNAs in plant-virus interactions. Based on the abundance of siRNAs and lack of PPV virus accumulation in transgenic plants harboring UTR/P1 and CP hairpin construct, we have concluded that UTR/P1 and CP are likely the best viral regions for induction of resistance against PPV.