The Mighty Bt: Interactions between pests and pesticidal proteins from Bacillus thuringiensis

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Ferré Manzanero, Juan
Hernández Martínez, Patricia
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Bacillus thuringiensis (Bt) is one of the most popular biological pest control alternatives to the use of chemical insecticides. It can produce several insecticidal proteins that are used in Bt-based products or genetically modified crops, mainly Cry and Vip3 proteins. However, field-evolved resistance can threat its long-term use. It is a key point to understand how Bt proteins produce their toxicity inside the insect. In Chapters 1 and 2, we investigated the role of the ABCC2 transporter as receptor from Spodoptera exigua, a global polyphagous pest. Our results show the co-existence of two different sites in the SeABCC2, depending on which domain of the Cry proteins is binding. Also, the ability of Cry1A proteins to hetero-oligomerize was characterized. In Chapter 3, we characterized other ABC transporters from Heliothis virescens, identifying 2 novel transporters. To understand if any of the new ABCCs are involved in the toxicity of Bt proteins, we performed independent knock-outs of the ABC genes in eggs. Results from bioassays showed no difference in toxicity levels, pointing out that both ABCs do not have a marked role in toxicity. In Chapter 4, we fully characterized the truncated ABCC2 from a resistant colony of S. exigua and we explored if its mutations are affecting the functionality as a receptor for Cry1A proteins. By expressing the truncated version of the transporter, we found that the Cry1Ac protein could bind to it, indicating that the mutations do not affect the binding. Cell viability assays demonstrated that the Cry1A proteins tested exert toxicity, confirming its functionality. In Chapter 5, we analyzed an O. furnacalis colony resistant to Cry1Ab. We explored the cross-resistance with other Cry1’s by performing bioassays, finding moderate-to-high levels of resistance to Cry1Aa, Cry1Ac, and Cry1F. We also performed binding assays, finding that the specific binding of Cry1Ab and Cry1Aa was reduced in the resistant colony. In Chapter 6, we studied the Vip3Aa-resistance of a H. virescens colony. Binding and ligand blot assays showed no significant alterations, indicating an alternative Vip3A-resistance mechanism to the alteration of binding sites in this colony. Lastly, in Chapter 7, we gathered all the available research on response mechanisms of insects to overcome the infection of Bt and its proteins. This review classifies these mechanisms according to which step of the mode of action is interfering with, in 8 categories: (Ingestion, Crystal solubilization, Activation, Toxin sequestration…) and shows how the complexity of the insect defense should be a matter of study in the Bt research world to ensure its long-term use. The results obtained here allow to understand some of the interactions between insects, Bt and its insecticidal proteins, which may be useful to guarantee the long-term use of this powerful organism.
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