Mechanism of action of Cry1ac toxin from Bacillus thuringiensis in Helicoverpa armigera (Lepidoptera: Noctuidae)

• Ano de defesa: 2021
• Autor(a) principal: Silva, Igor Henrique Sena da [UNESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Estadual Paulista (Unesp)
• Palavras-chave em Português: Lagarta Helicoverpa; Toxinas Cry; Mecanismo de ação; Manejo de resistência de insetos; Bacillus thuringiensis; Cotton bollworm; Cry toxins; Mechanism of action; Resistance management; Cadherin; Prohibitin
• Link de acesso: http://hdl.handle.net/11449/204912

- Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), known as Cotton bollworm is an insect pest of global importance in cotton crop. Nonetheless, it also attacks several other important economic crops worldwide, such as soybeans, corn, sorghum, wheat, beans, tomatoes, and ornamental plants. This insect is susceptible to some insecticidal Cry toxins from Bacillus thuringiensis (Bt) expressed in transgenic plants (Bt plants) or used in biopesticides. However, the capacity to evolve resistance to Bt plants have been threaten the technology in the long term. The most common mechanisms of resistance of lepidopterans to Cry toxins are mutations linked to Cry toxins receptors resulting in reduced binding. Thus, the identification and characterization of the putative Cry receptors is fundamental to better understand the mode of action of Cry toxins, retarding resistance evolution and producing Cry toxins more effective against insect pests. Previously, we identified H. armigera prohibitin (PHB) as a Cry1Ac-binding protein. The aim of this work was to further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), a midgut protein (MP) well-recognized as Cry1Ac-receptor. In addition, to characterize the interaction of those two MP with different Cry1A toxins. In this way, HaPHB-2 midgut protein and HaCAD toxin binding region fragment (TBR) from H. armigera were expressed in Escherichia coli cells and qualitative and quantitative binding assays with different Cry1 toxins were performed, as well as competition assays. We demonstrated that Cry1Ab, Cry1Ac and Cry1Fa toxins bound to HaPHB-2 similarly as to HaCAD-TBR. HaPHB-2 protein competed with Cry1Ac binding to H. armigera BBMVs. A reduction of toxin binding to HaBBMV was observed in the presence of HaPHB-2 in a concentration dependent way. Furthermore, different Cry1Ab mutant toxins located in domain II (Cry1Ab-F371A and Cry1Ab-G439D) or domain III (Cry1Ab-L511A and Cry1Ab-N514A), that were previously characterized to be affected in receptor binding, were analyzed regarding to their binding interaction with HaPHB-2 and toxicity against H. armigera. One β-16 mutant (Cry1Ab-N514A) showed increased binding to HaPHB-2 that correlated with six-fold higher toxicity against H. armigera while the other β-16 mutant (Cry1Ab-L511A) that was affected in binding to HaPHB-2 lost toxicity against H. armigera. We have found that the β-16 region from domain III of Cry1Ab is involved in interaction with HaPHB-2 and toxicity. This work identified a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect suggesting that this midgut protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigera. This is the first characterization of HaPHB-Cry1A interaction, supporting that HaPHB-2 also participates in the mechanism of action of Cry1Ab toxin in H. armigera, presenting new insights of the mode of action of Cry1 toxins in this important global pest.