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 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.
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This work was supported by National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering R01EB022376, DARPA HR0011-11-2-0003, DARPA N66001-12-C-4207, the Howard Hughes Medical Institute, and the US Department of Agriculture National Institute of Food and Agriculture and Agricultural Research Service Biotechnology Risk Assessment Grant Program 2012-33522-19791. A.H.B. was supported by the Harvard Chemical Biology Program and a National Science Foundation Graduate Research Fellowship. We are grateful to J. Carlson, J. Nageotte, D. Rappoli, J.-L. Kouadio, M. Zheng, J. Milligan, M. Huang, Z. Du, X. Zhou, E. Kraft, and J. Wang for their assistance.
The authors have filed a provisional patent application on the PACE system and related improvements.
Extended data figures and tables
a, Plasmids encoding an IPTG-inducible λ cI–SH2 cassette (‘DBD’) and an ATc-inducible activator-HA4 cassette (‘activator’) were co-transformed into the E. coli S1030 host strain and induced using either or both small molecules. T4 AsiA-mediated transcriptional activation required low-level expression of the σ70 (R541C/F563Y/L607P) mutant to alleviate AsiA toxicity. Use of RpoZ as the activation domain showed the greatest degree of transcriptional activation (~17-fold). b, DNA-binding domain variation shows that multivalent phage repressors yield a greater degree of transcriptional activation than the monomeric zinc finger Zif268. c, Transcriptional activation from a combination of the λcI DNA binding domain and RpoZ transcriptional activator was evaluated using several previously evolved protein–protein interactions involving either monobodies or DARPins, showing the generality of binding interaction detection. Error bars, s.d. of at least three independent biological replicates.
Extended Data Figure 2 Optimization of the PlacZ promoter for improved sensitivity and dynamic range.
a, Promoter and DNA-binding domain combinations tested during PlacZ optimization, showing uninduced and induced levels of absorbance-normalized luminescence. The SH2/HA4 interaction pair was used in all cases. The fold activation in each case was calculated as the ratio of the induced and uninduced luciferase expression signals. b, Graphical representation of the data in a, showing the wide distribution of promoter background levels and degrees of transcriptional activation. In a and b, the red and green dots indicate the starting (Plac62) and final (PlacZ-opt) promoter/DNA-binding domain combinations, respectively. Each data point in b reflects the average of at least three independent biological replicates.
a, Inducer titration of the interacting fusion proteins driving the two-hyrbid system. The black and green lines represent the uninduced (0 μM IPTG) and induced (1 μM IPTG) levels of IPTG-inducible 434cI–SH2 expression, while ATc induces expression of the rpoZ–HA4 cassette. In subsequent graphs and assays, the expression level resulting from the IPTG-inducible Plac promoter was measured by western blot and approximated using a constitutive promoter to reduce experimental variability. b, Degree of transcriptional activation using HA4 monobody mutants correlated with known binding affinities. The highest levels of activation resulted from Kd = low nanomolar affinities, while weak affinities in the Kd = low micromolar range could still be detected. c, Relationship between DNA-binding domain multivalency state (monomeric, dimeric, or tetrameric DNA-binding domain fused to the SH2 domain) and transcriptional activation resulting from the SH2/HA4 interaction, with higher multivalency states yielding greater activation levels. d, RBS modification enables robust modulation of the relative activation levels from the PlacZ-opt promoter using the SH2/HA4 interaction. e, Operator–promoter binding site spacing strongly affects transcriptional activation levels; 434cI binding at 61 base pairs upstream of the PlacZ-opt promoter resulted in the most robust activation. f, Linker extension to include one, two, or three G4S motifs result in reduced activation levels using the SH2/HA4 interacting pair. g, Phage plaque formation as a function of target protein multivalency. ‘No operator’ indicates a scrambled 434cI operator control accessory plasmid; ‘phage control’ indicates an accessory plasmid in which the phage shock promoter (activated by phage infection) drives gene III expression. h, Co-crystal structure of the ABL1 SH2 (blue) bound to the HA4 monobody (red), highlighting the interaction of HA4 Y87 (red spheres) with key residues of the phosophotyrosine-binding pocket (blue spheres) of the SH2 domain (Protein Data Bank accession number 3K2M). The phosphate ion is shown in orange at the interaction interface. i, Apparent binding activity of mutants of the HA4 monobody at position 87. Tyrosine, tryptophan, and phenylalanine are tolerated at position 87 and enable protein–protein interaction by bacterial two-hybrid assay. Error bars, s.d. of at least three independent biological replicates.
a, Protein sequence alignment of known Cry1Ac-binding motifs from cadherin receptors in several lepidopteran species, as well as the cadherin receptor from T. ni (TnCAD). The toxin-binding region (TBR; shown in red) of the known Cry1Ac-binding motifs differs from TnCAD at seven positions (shown in blue). Mutation of three residues in the TnCAD TBR (M1433F, L1436S, and D1437A) to resemble the corresponding positions of the cadherin-receptor TBRs yielded the evolutionary stepping-stone target TnTBR3. b, Schematic representations of the Cry1Ac and T. ni TBR3/CAD full-length receptors and fragments tested in this study. The red stars in the TnTBR3 variants represent the three mutations introduced into TnCAD to generate TnTBR3. c, Transcriptional activation assay using Cry1Ac and TnTBR3 fragments shows that the greatest degree of transcriptional activation resulted from full-length Cry1Ac together with TBR3 fragment 3 (TnTBR3-F3). RpoZ–Cry1Ac and 434cI–TnTBR3 fusions were used in all cases. d, Overnight phage enrichment assays using selection phages that encode either kanamycin resistance (KanR) only or KanR together with RpoZ–Cry1Ac. Compared with the KanR-only selection phage, the RpoZ–Cry1Ac selection phage enriches >26,000-fold overnight. e, Continuous propagation assays in the PACE format using either the KanR-only selection phage or the RpoZ–Cry1Ac selection phage show that the moderate affinity of Cry1Ac for TnTBR3 allows phage propagation at low flow rates (≤1.5 lagoon volumes per hour).
Extended Data Figure 5 Single-clone sequencing and evolved Cry1Ac characterization after PACE using the bacterial two-hybrid luminescence reporter.
a, Coding mutations of the tested RpoZ–Cry1Ac clones at the end of each of the four segments of PACE. Consensus mutations are coloured according to the segment in which they became highly enriched in the population (Fig. 3a). Mutations coloured in black were observed at low abundance (≤5% of sequenced clones). b, Mutational dissection of the consensus mutations from the first segment of PACE reveals the requirement for both D384Y and S404C to achieve high-level transcriptional activation using the TnTBR3-F3 target. Mutations listed in red occurred in the RpoZ activation domain, whereas mutations listed in blue occurred in the Cry1Ac domain. Error bars, s.d. of at least three independent biological replicates. c, Structure of wild-type Cry1Ac (Protein Data Bank accession number 4ARX) showing the positions of the evolved consensus mutations. The colours correspond to the PACE segments shown in Fig. 3 during which the mutations became highly abundant.
The number of reads mapped to the wild-type rpoZ–Cry1Ac reference sequence using (a) Pacific Biosciences (PacBio) or (b) Illumina sequencing. Time points are coloured according to the corresponding segment of the PACE experiment (Fig. 3a). c, In general, most PacBio reads aligned to the wt rpoZ–Cry1Ac reference sequence were found to cluster around ~2,200 base pairs, corresponding to the size of the full-length fusion gene and indicating high-quality sequencing reads. d, Illumina high-throughput sequencing yielded several high-quality single nucleotide polymorphisms across all time points. The corresponding mutations are shown in e.
Extended Data Figure 7 Insect diet bioassay activity of PACE-evolved Cry1Ac variants against various agricultural pests.
Two consensus and three stabilized PACE-evolved Cry1Ac variants were tested for activity in eleven pests: a, C. includes (soybean looper); b, Heliothis virescens (tobacco budworm); c, Helicoverpa zea (corn earworm); d, Plutella xylostella (diamondback moth); e, Agrotis ipsilon (black cutworm); f, Spodoptera frugiperda (fall armyworm); g, Anticarsia gemmatalis (velvetbean caterpillar); h, Diatraea saccharalis (sugarcane borer); Spodoptera eridania (southern armyworm); Leptinotarsa decemlineata (Colorado potato beetle); and Lygus lineolaris (tarnished plant bug). Stabilized variants showed enhanced activity in C. includens and H. virescens compared with wild-type Cry1Ac, and comparable activity to wild-type Cry1Ac in H. zea, P. xylostella, A. ipsilon, S. frugiperda, A. gemmatalis, and D. saccharalis. No activity was observed for any of the Cry1Ac variants at any tested dose for S. eridania, L. decemlineata, or L. lineolaris. No insect larvae mortality was observed for S. frugiperda, although high toxin doses greatly stunted growth.
The percentage sequence identity using the full-length cadherin receptor (a) or fragment used for directed evolution experiments (b) for insects tested in Extended Data Fig. 7. Numbers in parentheses denote the number of identical amino acids between the two receptors. In general, mortality and stunting data from diet bioassays correlate with cadherin receptor sequence identity.
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Badran, A., Guzov, V., Huai, Q. et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature 533, 58–63 (2016). https://doi.org/10.1038/nature17938
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