Sequence optimization and multiple gene-targeting improve the inhibitory efficacy of exogenous double-stranded RNA against pepper mottle virus in Nicotiana benthamiana

Abstract

Double-stranded RNA (dsRNA)-induced RNA interference is a promising agricultural technology for crop protection against various pathogens. Recent advances in this field have enhanced the overall efficiency with which this approach inhibits pathogenic viruses. Our previous study verified that treatment of Nicotiana benthamiana plants with dsRNAs targeting helper component-proteinase (HC-Pro) and nuclear inclusion b (NIb) genes protected the plant from pepper mottle virus (PepMoV) infection. The aim of this study was to improve the inhibitory efficacy of dsRNAs by optimizing the target sequences and their length and by targeting multiple genes via co-treatment of dsRNAs. Each of the two targeting dsRNAs were divided into three shorter compartments and we found that HC-Pro:mid-1st and NIb:mid-3rd showed significantly superior antiviral potency than the other fragments, including the parent dsRNA. In addition, we confirmed that the co-treatment of two dsRNAs targeting HC-Pro and NIb produced a greater inhibition of PepMoV replication than that obtained from individual dsRNA treatment. Complementing our previous study, this study will provide future directions for designing dsRNAs and enhancing their efficiency in dsRNA-mediated RNA interference technologies.