Short hairpin RNA engineering: In planta gene silencing of potato virus Y

RNA interference is a defense response against virus multiplication. The goal of this paper was to test if expressing virus-specific shRNAs against potato virus Y (PVY) could confer resistance against PVY in potatoes. The recombinant binary vectors consisted of short hairpin RNA (shRNA) transgenes, expressed under the CaMV35S promoter, directed against the conserved portion of the potato virus Y (PVY) coat protein gene. To provide the shRNA a particular shape and stability in vivo, stem and loop sequences from a microRNA (miR403; an active regulatory microRNA in potato) were included with the siRNA sequences. The shRNA constructs were delivered into the Cardinal potato variety using Agrobacterium, and transgenic status was confirmed by polymerase chain reaction (PCR) and Southern blot. Compared to wild-type plants inoculated with PVY, expression of PVY coat protein mRNA, as assayed by reverse transcriptase (RT)-PCR, was reduced to 0.05% in shRNA1-transgenic potato lines and 22% in shRNA4-transgenic lines. In addition, via northern blots, the maximum accumulation of the transgene-specific siRNA was observed in shRNA1 transgenic plants, less was observed in shRNA4-transgenic lines, and the least amount was detected in control plants, which correlates with silencing of the PVY coat protein gene. Conclusively, by simultaneous RNA silencing, shRNA1-transgenic plants were found to be almost immune to PVY infection compared with shRNA4-potato plants. Therefore, the expression of virus specific shRNAs is a novel, effective, and predictable approach to engineer resistance against PVY. (C) 2016 Elsevier Ltd. All rights reserved.