RNA interference in the tobacco hornworm, manduca sexta, using plastid-encoded long double-stranded RNA
Citation
Burke, W.G., Kaplanoglu, E., Kolotilin, I., Menassa, R., Donly, C. (2019). RNA interference in the tobacco hornworm, manduca sexta, using plastid-encoded long double-stranded RNA. Frontiers in Plant Science, [online] 10 http://dx.doi.org/10.3389/fpls.2019.00313
Plain language summary
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 insects are resistant to this process. In this study, we set out to test a strategy that uses a plants own chloroplasts to produce the molecules needed for RNAi, called double-stranded RNA (dsRNA), to achieve RNAi in one such insect, the tobacco hornworm. Using a hornworm gene called v-ATPaseA as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the dsRNAs to hornworm larvae. We found that a long dsRNA was the most effective in killing the insects and silencing the target gene when delivered to the insects orally in a water droplet. Next we transformed the chloroplast genome of the hornworm host plant, Nicotiana tabacum, to produce this product in its plastids and performed bioassays with hornworm larvae on the resulting plants. However, we found that when produced in the chloroplasts, the dsRNA had no effect on insect survival and no statistically significant effect on expression of the target gene. We also found that there was less of the long dsRNA present in the chloroplasts than if a short dsRNA was used. 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 one of the factors that affects effectiveness of RNAi driven by dsRNA made in plants, is the length of the product. This information will be important in designing the next generation of insect protection for crops based on RNAi.
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.