Barra, Eleonora (2021) Use of RNAi to develop new biotechnologies for insect control. [Tesi di dottorato]

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The identification of new bioinsecticides and of their efficient delivery strategies is one of the current approaches to reduce the use of synthetic chemicals in agriculture. The use of natural antagonists as a source of virulence factors or of molecular technologies that mimic their negative effect on the host insects paves the way toward the development of new bioinspired tools of pest control. To this aim, RNA interference (RNAi) can be used to artificially down-regulate host genes negatively targeted by virulence factors of natural antagonists, providing new opportunities for pest control. Recently, it has been demonstrated that RNAi-mediated silencing of an immune gene (Sl 102), to reproduce the negative effect of a polydnavirus associated with a parasitic wasp, generates an immunosuppressed phenotype in Spodoptera littoralis (Lepidoptera: Noctuidae) larvae, making them more susceptible to the entomopathogen Bacillus thuringiensis. To exploit this novel pest control tool, it is essential to limit the environmental and insect gut degradation of double strand RNA (dsRNA) molecules. Here we contribute to this research goal by developing two delivery strategies, based on the expression of dsRNA molecules in Escherichia coli and transgenic tobacco plants. Experimental larvae ingesting bacteria or plant tissue expressing Sl 102 dsRNA showed marked transcriptional down-regulation of the targeted gene and both enhanced the killing activity of a Bt-based biopesticide (Xentari™), demonstrating that these two delivery strategies were both effective. Moreover, to further enhance the immunosuppression and the resulting biocontrol level by Bt, we concurrently silenced an additional immune gene (Sl gasmin), which encodes a protein acting as an opsonizing factor promoting phagocytosis. The double silencing was successfully observed and was associated with a significant impairment of both encapsulation/nodulation and phagocytosis. However, this double immune deficiency did not induce a synergistic response, further enhancing the killing activity by Bt, which, indeed, was similar to that observed when only Sl 102 gene was silenced. In conclusion, bacteria and transgenic plants expressing Sl 102 dsRNA appear to be two promising delivery strategies for field application. Their use to induce immunosuppression offers the possibility to pursue a new bio-inspired strategy to suppress pests based on the enhancement of their sensitivity to natural antagonists and, thus, reinforcing the important ecosystem service they provide.

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