Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein–protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant K d = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. Phage-assisted continuous evolution (PACE) rapidly evolves Bacillus thuringiensis toxins through more than 500 generations of mutation, selection, and replication to bind a receptor expressed on the surface of insect-pest midgut cells. Beating Bt resistance in insect pests The emergence of insects resistant to Bacillus thuringiensis δ-endotoxins (Bt toxins) is threatening to reduce the effectiveness of this system in crops engineered to carry these insecticidal proteins. David Liu and colleagues have used phage-assisted continuous evolution (PACE) selection to rapidly evolve high-affinity protein–protein interactions, and applied the system to evolve Bt toxin variants that kill insects through binding a new insect gut cell protein target — a cadherin-like receptor from the insect pest Trichoplusia ni . The modified Bt toxins are shown to overcome Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.