Generation of PVY Coat Protein siRNAs in Transgenic Potatoes Resistant to PVY

Global climate change has the potential to quickly alter the distribution and profile of crop pests and pathogens. Ready tools for mobilization of resistance into elite crops will be part of an integrated strategy to maintain agricultural productivity. Engineered virus resistance through expression of coat protein is well established. The mechanisms of virus suppression by small RNAs (sRNAs) have only recently been elucidated. Understanding the role of sRNAs in gene regulation and development is rapidly evolving. High throughput sequencing (HTS) can generate large data sets of sRNA sequences which can be analyzed to recognize the abundant sRNAs that result from transcript degradation and to identify the regulatory micro RNAs (miRNA) involved in gene regulation and small inhibitory RNAs (siRNA) that confer virus resistance. Transgenic Solanum tuberosum that are glass house or field grown and that express the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) from the 409s promoter with GBSS6 intron as a spacer exhibited resistance to PVY. Southern blots of genomic DNA indicated one or two copies of the transgene and analysis of HTS data of the small RNA population indicated high levels of siRNA production from the transgenic hairpin construct. Up to 47 of the top 200 most frequent sRNA reads were attributable to the PVY-CP transgenic construct in Ranger Russet line #5. This combination of transgene and transcription strength is functional for constructing efficient resistance cassettes for crop protection. Up to 95 of the top 200 sRNA sequences were 21 nucleotides in length with as many as 19 sequences corresponding to known miRNA transcripts. Additional endogenous sRNAs with homology to resistance gene analogs may be indicative of additional or complimentary resistance mechanisms produced in concert with the PVY siRNAs arising from PVY-CP RNA transcription.