Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis

Abstract

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults

“…To amplify cDNA encoding a putative Lgl protein from D. v. virgifera , specific primers were designed based on transcriptome data from D. v. virgifera . Overlapping PCR products were then amplified with Sanger sequencing as described in Cooper, Song, Shi et al (2020). Finally, specific primers designed near the start and stop codon were used to amplify the entire open reading frame (ORF) and confirm the full‐length sequence (Table ).…”

“…To verify the identity of the putative Lgl transcript, multiple sequence alignments, phylogenetic analyses, domain comparisons, and subcellular localization predictions were performed as described in Cooper, Song, Shi et al (2020). Similar analyses were performed on Lgl from O. nubilalis (MT467568) and T. castaneum (MT467569) for comparison with DvLgl.…”

“…Tissue‐specific and developmental stage‐specific expression profiles were generated as described in Cooper, Song, Shi et al (2020). The developmental stages analyzed for D. v. virgifera included: eggs; first‐, second‐, and third‐instar larvae; pupae; adult males; and adult females.…”

“…Reverse transcription‐quantitative PCR (RT‐qPCR) was used to examine tissue and developmental stage‐specific expression of DvLgl and OnLgl and examine RNAi‐mediated suppression of DvLgl after ds DvLgl feeding in D. v. virgifera . RT‐qPCR was performed following the minimum information necessary for RT‐qPCR (MIQE) guidelines (Bustin et al, 2009) as described in Cooper, Yu, Biondi et al (2020), and Cooper, Song, Shi et al, (2020). Ribosomal protein 3S ( DvRPS3 ) was used for normalization of relative expression across D. v. virgifera samples, and the geometric mean of OnRPS3 and OnEF1α was used for normalization of relative expression among O. nubilalis samples.…”

“…Coleopterans, like D. v. virgifera , are well known for being highly amenable to RNAi, whereas lepidopterans typically are not (Cooper et al, 2019; Terenius et al, 2011; Zhu & Palli, 2020). Therefore, it is hard to know if the lack of phenotype recently observed after the suppression of Lgl in multiple developmental stages of O. nubialis is due to variation in the susceptibility of this gene to RNAi in insects (Cooper, Song, Yu, et al, 2021), due to logistical differences in experimental design (Cooper et al, 2019; Terenius et al, 2011), or due to another mechanism of low RNAi efficiency in O. nubialis (Cooper et al, 2019, Cooper, Song, Shi et al, 2020, Cooper, Yu, Biondi et al, 2020; Cooper, Song, Shi, et al, 2021; Cooper, Song, Yu, et al, 2021). Therefore, identification and characterization of Lgl in D. v. virgifera may clarify the suitability of Lgl as a target or reporter gene for RNAi in insects.…”