Tidings from Bacillus thuringiensis: exploring new targets, enhancing potency, and unraveling response mechanisms

Ayudas para contratos predoctorales para la formación de doctores. BES-2016-079134

The biopesticide market is growing due to the increasing demand for sustainable agriculture and target-specific alternatives to synthetic pesticides. Bacillus thuringiensis (Bt) is one of the most important biopesticides producing vegetative insecticidal proteins (Vip) and crystal proteins (Cry). However, its effectiveness is limited by a narrow host range and emerging resistance in target insect populations. To overcome these limitations, efforts are underway to isolate new Bt strains that produce novel toxins with an expanded spectrum of activity to control existing and emerging pests; besides, synergistic compounds have been used to potentiate activity and delay resistance. Hence, the first objective of the thesis was to evaluate and optimize the insecticidal properties of Bt, which is tackled in Chapter I and II. In Chapter I, the strain HD-537 was characterized and tested for potential use in controlling Blatella germanica. Our results showed that HD-537 produced Cry1 and Cry2 proteins. An oral bioassay was set up employing a cotton-covered glass bottle as a toxin dispenser. After 10 days of treatment, no effect on survival rate or growth was observed. Thus, HD-537 crystal proteins are not effective as biological control agents against B. germanica. Chapter II aimed to broaden the spectrum and potentiate activity of Cry1Aa, Cry1Ab, Cry1Ca, Cry1Ia and Vip3Aa against Grapholita molesta and Spodoptera exigua employing a cadherin fragment from S. exigua (rSeCad1bp) and another from Tenebrio molitor (rTmCad1p). Single-dose assays showed a singular enhancement in S. exigua (Cry1Ia:rSeCad1bp) and three in G. molesta (Cry1Aa:rSeCad1bp, Vip3Aa:rSeCad1bp and Cry1Aa:rTmCad1p). Dose-response assays revealed that mixing Cry1Aa and Cry1Ab with rSeCad1bp increased their toxicity to G. molesta by 2.6 and 2.8- fold, respectively. Therefore, our work found that rSeCad1bp is a remarkable candidate to synergize Cry1Aa and Cry1Ab in G. molesta. Furthermore, based on our results, we proposed that Cry-Cadherin interaction, protein stability enhancement or promotion of oligomer formation might not be sufficient for boosting Cry1A toxicity. Instead, cadherin-mediated enhancement of Cry1Aa toxicity in G. molesta might be linked with an increase in binding to BBMV. In contrast, no correlation was observed for Cry1Ab. Hence, alternative mechanisms may be involved in the synergism produced by rSeCad1bp on Cry1Ab toxicity towards G. molesta. Bt challenge triggers a complex host-intrinsic cellular defense mechanism, including autophagy, to counteract bacterial damage. Alteration of these response mechanisms might influence host susceptibility. In Chapter III, we assessed the second objective of the thesis: the response mechanism of Bombyx mori to Cry1Aa from an autophagic perspective. Our analysis revealed that, upon B. mori intoxication with Cry1Aa at sublethal and lethal doses for 48 hours, ATG8-PE increased in a dose-independent manner. However, no induction of ATG8 gene or increase in acid phosphatase activity was detected. Consequently, two possible scenarios might be suggested: one in which Cry1Aa inhibits autophagy, and another in which Cry1Aa induces ATG8-dependent secretory pathways. Both represent novel perspectives on the Cry host response. In conclusion, the findings presented in the thesis provide a foundation for developing next-generation biopesticides, thereby contributing to the future of Bt-based pest control strategies.

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