Abstract
Insecticidal toxins from Bacillus thuringiensis (Bt) have been extensively and successfully used in genetically engineered crops for decades but continue to face challenges from the adaptive resistance in the insect population. The Bt binary toxin Vip1Ad1(Vip1) and Vip2Ag1(Vip2), a promising next-generation candidate gene combination for transgenic crops, have demonstrated high efficacy against the destructive coleopteran pest white grubs, however, their mode of action remains largely elusive. In this study, we report cryo-EM structures of the heptameric Vip1-pore and Vip2-bound Vip1-pore complex, capturing a series of putative assembly-related intermediates that suggest a binary toxin assembly and translocation pathway. Together with structure-guided mutagenesis, these data provide insights into a sequential assembly of binary complex and a sequence-independent translocation mechanism. Proof-of-principle experiments showed successful delivery of a desired protein cargo into host cells based on the mini-Vip2-Vip1 pore system, paving the way for developing much needed extracellular pesticidal protein delivery platforms. These findings not only clarify the assembly and translocation mechanism of the binary insecticidal toxin pair but also offer an excellent alternative model to investigate human-pathogenic pore-forming toxins because of its similarity and biosafety.
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Data availability
Cryo-EM maps were deposited to the Electron Microscopy Data Bank (https://www.ebi.ac.uk/emdb/) under accession codes EMD-65010 (Vip1 heptameric pore), EMD-65009 (Vip2 bound heptameric pore complex), EMD-65007 (1:5 (Vip2:Vip1) complex). The corresponding coordinates were deposited to the Protein Data Bank (https://www.rcsb.org/) under accession codes 9VEK (Vip1 heptameric pore), 9VEJ (Vip2 bound heptameric pore complex), 9VEG (1:5 (Vip2:Vip1) complex). All other published PDB codes cited in this paper are 6SMS, 7YVQ and 1QS1. All data generated or analyzed during this study are included in this article. Source data are provided as a Source Data file. Source data are provided with this paper.
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Acknowledgements
We would like to thank Ms. Danyang Li and Xiangning Li from the Core Facility of Wuhan. University for their assistance with the cryo-EM data collection, thank Prof. Changyou Li and Dr. Guiling Zheng from Qingdao Agricultural University to supply H.parallela HP9-2 Cells, thank Prof. Qing Yang, Prof. Changlong Shu and Dr. Lili Geng from Institute of Plant Protection, Chinese Academy of Agricultural Sciences for their technical guidance about the insecticidal bioassay. We thank Dr. Qing Chang for providing AUC support at Biomolecule Preparation and Characterization Platform of the Technology Center for Protein Research, Tsinghua University. We thank Derek Duong, Dr. Linfeng Gao, and Dr. Baohua Chen for improving the language and style of the manuscript. This work is supported by grants from Natural Science Foundation of Wuhan (grant number 2024040801020228) for Z.L., Provincial Natural Science Foundation of China (grant number 2024AFB026) for Z.L., a startup fund from Wuhan University awarded to P.C., and Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSCB-202401 and CAAS-ZDRW202304 to Z.W.).
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Conceptualization: T.Z. and P.C. Methodology: T.Z., J.R., L.L., Z.L., and Z.W. Investigation: T.Z., J.R., L.L., Z.W., and X.H. Writing: T.Z., Z.W., and P.C. Supervision: J.Z. and P.C. Funding acquisition: Z.L., Z.W., and P.C.
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Zhao, T., Wang, Z., Ren, J. et al. Structural basis for the assembly and translocation of the Vip1-Vip2 insecticidal toxin from Bacillus thuringiensis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71211-7
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DOI: https://doi.org/10.1038/s41467-026-71211-7
