RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA

Abstract

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.

Strategies for enhancing the efficiency of RNA interference in insects

“…First proposed in 2007 through modification of corn for control of D.v. virgifera , 7 genetic engineering has since been used in a variety of organisms, including microbes (fungi and bacteria), 47,48 viruses, 49 plants, 50 and even microalgae 51 . The organisms have been engineered either to synthesize dsRNA that will be ingested by the target pest insect (bacteria, fungi, plants, microalgae) or to carry the genetic instructions for synthesizing target dsRNAs in either host plants or target insects (viruses).…”

Insights Into Genetic and Molecular Elements for Transgenic Crop Development

“…However, the dynamics of dsRNA processing and siRNA production in insects are somewhat different from those in plants (although these are still not well understood). Thus, siRNAs (20-24 nt in length) produced in planta are not efficient for knocking out insect genes, while full-length dsRNAs have been shown to be more efficient (Whyard, 2015;Burke et al, 2019). Burke et al (2019) showed that long dsRNAs in planta produced from plastid transformation and targeting the v-ATPaseA gene of Manduca sexta led to inefficient RNAi-based insect control, suggesting that the stability and length of the dsRNA may have been affected.…”

“…Thus, siRNAs (20-24 nt in length) produced in planta are not efficient for knocking out insect genes, while full-length dsRNAs have been shown to be more efficient (Whyard, 2015;Burke et al, 2019). Burke et al (2019) showed that long dsRNAs in planta produced from plastid transformation and targeting the v-ATPaseA gene of Manduca sexta led to inefficient RNAi-based insect control, suggesting that the stability and length of the dsRNA may have been affected. To overcome this problem, long dsRNA molecules were engineered in planta with a viroid-like structure to knock out insect pest genes.…”

Tobacco Hornworm (Manduca sexta) caterpillars as a novel host model for the study of fungal virulence and drug efficacy

“…M. sexta laboratory stocks have been derived from animals collected in North Carolina, USA [33] and have been maintained in laboratories on both sides of the Atlantic for several decades. M. sexta's research portfolio includes a draft genome sequence that has been complemented with tissue-specific transcriptomic analyses [34], numerous successful applications of RNAi [35][36][37][38][39]. Down-stream analyses [40] of the animal's innate immunity [41], which also comprises b-(1,3)-glucan recognition proteins [41], are facilitated by protocols for the efficient extraction of hemocytes.…”