Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm

PLoS One. 2021 Dec 20;16(12):e0260532. doi: 10.1371/journal.pone.0260532. eCollection 2021.

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel β-sheets organized into β-sandwich layers. The amino-terminal domains 1-3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long β-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4-6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacillus thuringiensis / metabolism*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Coleoptera / drug effects
  • Coleoptera / growth & development
  • Crystallography, X-Ray
  • Insecticides / chemistry
  • Insecticides / pharmacology*
  • Intestines / metabolism
  • Larva / drug effects
  • Larva / metabolism
  • Mutagenesis, Site-Directed
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Zea mays / metabolism
  • Zea mays / parasitology

Substances

  • Bacterial Proteins
  • Insecticides
  • Recombinant Proteins

Grants and funding

Bayer accepted no outside funding for the research presented in this manuscript. Bayer provided funding and reagents to MA and YP at Kansas State University and provided support in the form of salaries and research materials for authors J-LK, MZ, JZ, DC, DD, SD, JM, CT, PM, TR, CK, TP, YY, WM, KG, AJ, and JH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.