A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust. (July 2017) - Song, Huifang
Abstract: Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3 . Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3- suppressed larvae or control larvae injected with ds GFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2- suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with ds GFP, ds LmdsRNase2 or ds LmdsRNase3 and chitinase 10 ( LmCht10 ) or chitin synthase 1 ( LmCHS1 ) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with ds LmdsRNase2 (48% and 22%, for ds LmCht10 and ds LmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust. Graphical abstract: Highlights: We characterized cDNAs of two midgut-specific double-stranded RNA (dsRNA) degrading enzymes (dsRNases) from L. migratoria. dsRNA incubated in midgut fluid from LmdsRNase2- suppressed larvae was more stable. RNAi of LmCht10 and LmCHS1 after RNAi of LmdsRNase2 implicates LmdsRNase2 in reducing RNAi efficiency. Recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. LmdsRNase2 in the midgut is a major factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.