RNAi Pesticides: Toward Hornworm Control via Bacterial dsRNA




Robinson, Elizabeth C.

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Conventional chemical insecticides frequently have negative off-target effects on other organisms. Both important pollinators and human consumers have been harmed due to insecticide consumption. Some researchers have proposed using RNA interference (RNAi) to target essential genes that are specific to agricultural pests: if a pest insect ingests double-stranded RNA (dsRNA) with a sequence identical to an important gene, RNAi can reduce expression of that gene, thereby killing the pest. This could mitigate harmful effects on other insects or humans, because genes specific to one pest species can be targeted. However, dsRNA is unstable outside of living cells, and would quickly deteriorate if simply sprayed over a crop field. Instead, it may be possible to modify bacteria that naturally live on crop plant leaves to produce the dsRNA continuously. Here, I establish the necessary background for using this insecticide alternative against the tobacco hornworm, Manduca sexta. I confirm that a non-pathogenic strain of the common crop symbiont, Pseudomonas syringae (PSY), colonizes tomato leaves to high titers for at least 72 hours, suggesting that this species could remain effective at producing dsRNA for long periods of time. I construct two plasmids that express dsRNA for portions of the M. sexta vascular ATPase gene, which is essential to M. sexta survival, and transform them into two strains of PSY. I integrate a gene encoding expression of a green fluorescent protein (GFP) into both PSY genomes so that colonization can be tracked more easily. Finally, I attempt to replicate a study that increased tobacco hornworm mortality upon ingestion of vATPase dsRNA—however, this replication was unsuccessful, likely due to large numbers of M. sexta caterpillars dying of an infectious disease. Overall, these results support the concept that using symbiotic leaf bacteria to continuously produce and stabilize insecticidal dsRNA may be feasible. The tools I have developed suggest that trials on live pests and live plants may be just around the corner. In the future, the stability, sustainability, and cost effectiveness of dsRNA insecticides could allow us to protect crop plants without sacrificing human or environmental health.


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