CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti
Por:
Pacheco, S, Gallegos, AS, Peláez-Aguilar, AE, Sánchez, J, Gómez, I, Soberón, M, Bravo, A
Publicada:
1 jun 2024
Resumen:
The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins. Author summary The Cry toxins produced by the bacterium Bacillus thuringiensis subsp. israelensis have been used for decades to control populations of mosquito responsible for spreading febrile diseases in tropical regions. These insecticidal toxins specifically target the midgut cells of larvae. It is widely accepted that two membrane proteins, cadherin (Aae-Cad) and membrane-bound alkaline phosphatase (Aae-mALP), are receptors for Cry toxins in A. aegypti. With the aim to study the functional role of Aae-Cad and Aae-mALP in the mode of action of mosquitocidal Cry toxins, in this study we employed the CRISPR-Cas9 system to generate knockout A. aegypti strains deficient in either Aae-Cad or Aae-mALP. Interestingly, we found a sex-linked association for mutations in Aae-Cad generated by CRISPR-Cas9, attributable to its proximity to the sex-determining M-factor on chromosome 1. Furthermore, the CRISPR-Cas9-mediated knockout of Aae-Cad or Aae-mALP proteins did not affect the normal development of the insects. Surprisingly, we found that the absence of Aae-Cad in the KO-females or Aae-mALP KO A. aegypti did not generate a resistance phenotype to four different Cry toxins, Cry11Aa, Cry11Ba, Cry4Ba and Cry4Aa, indicating that additional molecules are required in the mode of action of Cry toxins.
Filiaciones:
Pacheco, S:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Gallegos, AS:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Peláez-Aguilar, AE:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Sánchez, J:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Gómez, I:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Soberón, M:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
Bravo, A:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Microbiol Mol, Cuernavaca, Morelos, Mexico
gold, All Open Access; Gold Open Access
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