Engineering Bacillus thuringiensis Cyt1Aa to function as a surrogate receptor of Cry1A lepidopteran insecticidal...

Transgenic crops expressing Bacillus thuringiensis (Bt) Cry1 insecticidal proteins is an efficient technology for the control of lepidopteran pests. However, practical resistance to Bt crops has been reported in 11 pest species, endangering the long-term efficacy of this technology. The insecticidal Cyt1Aa toxin is a versatile protein that synergizes the toxicity of mosquitocidal Cry proteins such as Cry11Aa or Cry4Ba by acting as a surrogate receptor that facilitates their oligomerization. Here, we engineered Cyt1AaA59C (a non-hemolytic variant of Cyt1Aa) to serve as a surrogate receptor of the lepidopteran Cry1Ab and Cry1Ac toxins, by introducing the complementarity-determining region, CDR3, amino acid sequence of the single-chain fragment variable antibody scFv73, that binds to Domain II loop 2 of Cry1A toxins, which induces Cry1A toxin oligomerization similarly to the larval gut cadherin or ABCC2 receptors. The scFv73 CDR3 amino acid sequence was inserted into three exposed loops of a Cyt1AaA59C, producing Cyt1Aa-73 hybrids. The three Cyt1Aa-73 hybrid proteins bound Cry1Ab and Cry1Ac in a similar manner as to the Manduca sexta cadherin fragment containing Cry1A binding site that induced oligomerization of Cry1Ab or Cry1Ac in vitro. In addition, Cyt1Aa-73 hybrid proteins enhanced the toxicity of these toxins against susceptible Plutella xylostella larvae. Furthermore, Cyt1Aa-73 L7 increased the toxicity of Cry1Ac against P. xylostella NO-QAGE strain, whose resistance to Cry1Ac is linked to a mutation on the ABCC2 transporter receptor. Our results demonstrates that Cyt1Aa can be engineered to function as surrogate receptor for other Cry proteins providing a potential novel tool to counter resistance to Bt crops.