Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance

The expression of insecticidal proteins from B. thuringiensis (Bt toxins) in crops has proved to be a valuable strategy for agricultural pest management1. Bt-toxin-producing crops have been widely adopted in agriculture with substantial economic and environmental benefits2, and have increased global agricultural productivity by an estimated US$78 billion from 1996 to 2013 (ref. 3). Unfortunately, Bt toxin resistance has evolved among insect pests and threatens the continued success of this strategy for pest control4. While resistance management strategies have been developed, including the use of multiple Bt toxins and preserving susceptible alleles in insect populations, the evolution of insect resistance to Bt toxins remains the most serious current threat to sustaining the gains offered by transgenic crops4. Bt toxins interact with protein receptors on the surface of insect midgut cells, leading to pore formation in the cell membrane and cell death5. Bt toxin resistance is commonly associated with the mutation, downregulation, or deletion of these receptors2. We hypothesized that it might be possible to overcome Bt toxin resistance by evolving novel Bt toxins that bind with high affinity to new gut cell receptor proteins in insects. If successful, such an approach has the potential to alter toxin specificity, improve toxin potency, and bypass receptor-related resistance mechanisms. Here we use phage-assisted continuous evolution (PACE) to rapidly evolve Bt toxins through more than 500 generations of mutation, selection, and replication to bind a new receptor expressed on the surface of insect midgut cells. PACE-derived Bt toxins bind the new receptor with high affinity and specificity, induce target receptor-dependent lysis of insect cells, and enhance the insecticidal activity against both sensitive and Bt-resistant insect larvae up to 335-fold. Collectively, these results establish an approach to overcoming Bt toxin resistance and provide a new platform for the rapid evolution of other proteinbinding biomolecules. Development of protein-binding PACE PACE has mediated the rapid laboratory evolution of diverse protein classes including polymerases, proteases, and genome-editing proteins, yielding variants with highly altered activities and specificities6–12. While PACE has not been previously used to evolve protein-binding activity, we speculated that the bacterial two-hybrid system13 could serve as the basis of a protein-binding PACE selection (Fig. 1a). Target binding results in localization of RNA polymerase upstream of a reporter gene, initiating gene expression. To adapt this system into a protein-binding selection for PACE, we envisioned that protein:target binding could instead activate the expression of the filamentous bacteriophage gene III, which is required for the infectivity of progeny phage6 (Fig. 1b). To maximize the sensitivity of the bacterial two-hybrid, we extensively optimized parameters including (1) transcriptional activation and DNA-binding domains, (2) protein expression level, (3) interaction binding affinity, (4) DNA-binding domain multivalency state, (5) reporter gene ribosome-binding site, (6) operator–promoter distance, (7) RNA polymerase–promoter affinity, and (8) DNAbinding domain–bait linker length. While the previously described bacterial two-hybrid system yielded a 17-fold increase in transcriptional activation using a model high-affinity interaction (HA4 monobody binding to the SH2 domain of ABL1 kinase)14, our optimized system enhanced transcriptional activation >200-fold using the same interaction (Extended Data Figs 1–3). This system consists of the Escherichia coli RNA polymerase omega subunit (RpoZ) as the activation domain, the 434 phage cI repressor as the DNA-binding domain, and an optimized PlacZ-derived promoter (PlacZ-opt) to drive reporter transcription. Together, these results extend and improve previously described bacterial systems13 that transduce protein-target binding into gene expression in a manner that can be tuned by the researcher. 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 Kd = 11–41 nM), kill TnCADexpressing 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.