Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera

Xiping Niu,Lisa Procyk,Albert Lu,Samantha Griffin,Claudia Perez-Ortega,Jun Zhang,Mark E Nelson,Deirdre M Kapka-Kitzman,Adane Kassa,James Hasler

doi:10.1038/s41598-020-72572-9

Xiping Niu, Lisa Procyk + Show 8 more

Open Access

https://doi.org/10.1038/s41598-020-72572-9

Copy DOI

Abstract

Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.

Highlights

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a key invasive insect pest of maize in the United States and Europe[1,2,3]
Dvv-isotig07787 or DvABCB2 is identical to the sequence found within a 20 cM genomic region that included multiple candidate genes linked to Cry3Bb1 resistance[49]
The identity of DvABCB1 to DvABCB2 is 67% which is lower than its identity to CtABCB1

Summary

Introduction

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a key invasive insect pest of maize in the United States and Europe[1,2,3]. Several protein families including the ATP-binding cassette (ABC) transporters, cadherin, aminopeptidase N (APN), and alkaline phosphatase (ALP) have been identified as receptors of Cry toxins in Lepidoptera where more extensive studies were p erformed[36,37] Some of these protein classes are differentially expressed in Cry3-resistant WCR or WCR treated with Bt toxins (Cry34/35Ab1, Cry3Bb1, or eCry3.1Ab)[35,38,39] but have yet to be validated as functional receptors. In Coleoptera, ABCB1was identified as a functional receptor for Cry3Aa in leaf beetle (Chrysomela tremula) using genetic linkage to resistance coupled with heterologous expression of the wild type gene to confer Cry3Aa sensitivity to Sf9 c ells[46] Based on these reports we identified a putative WCR orthologue, ABCB1, and the highly homologous sequence, ABCB2, to characterize and understand whether either or both serve similar roles in mediating Cry[3] toxicity in this important pest of maize. Reduced expression and alternative splicing of DvABCB1 transcripts were identified in mCry3A resistant WCR explaining the reduced binding of mCry3A that was previously reported[21]

Methods

Results

Conclusion