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. 2022 Feb 26:2:100033.
doi: 10.1016/j.cris.2022.100033. eCollection 2022.

Engineering of Cry3Bb1 provides mechanistic insights toward countering western corn rootworm resistance

Suyog S Kuwar  1 , Ruchir Mishra  1 , Rahul Banerjee  1 , Jason Milligan  2 , Timothy Rydel  2 , Zijin Du  2 , Zhidong Xie  2 , Sergey Ivashuta  2 , Jean-Louis Kouadio  2 , Jason M Meyer  2 , Bryony C Bonning  1
Affiliations

Engineering of Cry3Bb1 provides mechanistic insights toward countering western corn rootworm resistance

Suyog S Kuwar et al. Curr Res Insect Sci. .

Abstract

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

Keywords: Cry3Bb1; gut binding peptide; phage display; resistance; western corn rootworm.

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Conflict of interest statement

Bryony C. Bonning reports financial support was provided by Bayer Corp. Jason Milligan reports a relationship with Bayer Corp that includes: employment. Timothy Rydel reports a relationship with Bayer Corp that includes: employment. Zijin Du reports a relationship with Bayer Corp that includes: employment. Zhidong Xie reports a relationship with Bayer Corp that includes: employment. Sergey Ivashuta reports a relationship with Bayer Corp that includes: employment. Jean-Louis Kouadio reports a relationship with Bayer Corp that includes: employment. Jason M. Meyer reports a relationship with Bayer Corp that includes: employment.

Figures

Figure 1:
Figure 1
Workflow for protein engineering process in this study.
Figure 2:
Figure 2
Sites in Cry3Bb1 used for peptide insertion. The amino acid regions used for peptide insertion in domains II (D.II) and III (D.III) are indicated. For each region, the percentage of variants that retained activity against Cry3Bb1-susceptible WCR and the number produced and tested are indicated.
Figure 3:
Figure 3
Relative binding of native and variant Cry3Bb1 to s-WCR BBMV. Relative intensities of Cry3Bb1 bands in this representative western blot-pull down assay was quantified with FlourChem M FM0591 imaging software. Binding to BBMV was increased for a subset of variants relative to native Cry3Bb1. Variant 138 also showed increased binding in a single replicate (not shown).
See this image and copyright information in PMC

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