Deep sequencing of transgenic Nicotiana benthamiana plants, resistant to plum pox virus, and different potential of hairpin constructs in producing viral small RNAs

Abstract

Globally widespread of Plum pox virus (PPV) and the economic importance of stone fruits, as its natural host, signify the necessity and importance of PPV resistance programs. In the present study Agrobacterium-mediated transformation of Nicotiana benthamiana was performed using four inverted repeat constructs, derived from a PPV-T isolate; covering genomic regions: UTR/P1, HCPro, HCPro/P3 and CP; 752, 649, 594, 990 nucleotides, respectively. The efficiency of virus resistance was evaluated by inoculation of transgenic plants with PPV-D, -M, and -T strains. Aiming to compare the potential of hairpin structures in the production of siRNAs and miRNAs in both wild-type and transgenic plants was determined by small RNA high-throughput sequencing. siRNA analysis revealed various potentials of hairpin constructs in producing siRNAs and their accumulation in target regions. Expression profiles of several known and novel miRNAs are dramatically changed to PPV infection on both wild-type and transgenic plants, demonstrating plausible involvement of these miRNAs in plant-virus interaction progress. Based on the abundance of siRNAs and lack of PPV virus accumulation transgenic plants harboring UTR/P1 and CP hairpin construct, it is concluded that UTR/P1 and CP are likely to be the best viral region for induction of resistance.