Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm.

Laura Franz,Klaus Raming,Ralf Nauen

doi:10.3390/toxins14020157

Abstract

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.

Highlights

fall armyworm (FAW) pest management strategies largely rely on the use of synthetic insecticides and transgenic crops, e.g., corn or cotton expressing Bacillus thuringiensis (Bt) insecticidal proteins such as Cry and Vip toxins [11,12,13]
The objective of our study was to address this knowledge gap by functionally testing the interaction of several recombinantly expressed FAW ATP-binding cassette transporter C2 (ABCC2) variants mutated in extracellular loop 4 (ECL4) with Cry1F, Cry1A.105 and Cry1Ab
(3D) Cry toxin-mediated toxicity and resistance several lepidopteran species [29,32,33,49,50,51]. This is supported by resultsinobtained with ABCC2 species [29,32,33,49,50,51]. This is supported by results obtained with ABCC2 knockout knockout lines of S. frugiperda expressing high levels of resistance against Cry1F and lines of S. frugiperda expressing high levels of resistance against Cry1F and Cry1Ab [41]

Summary

Introduction

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), fall armyworm (FAW), is a highly-destructive moth pest native to the tropical and subtropical Americas that feeds on a broad range of host plants [1,2], including important row crops such as corn, cotton and soybean [3,4]. FAW recently invaded the Eastern Hemisphere and the first outbreaks were described in West Africa in 2016 [5], from where it rapidly dispersed to many countries in. FAW pest management strategies largely rely on the use of synthetic insecticides and transgenic crops, e.g., corn or cotton expressing Bacillus thuringiensis (Bt) insecticidal proteins such as Cry and Vip toxins [11,12,13]. Corn hybrids expressing Bt toxins remain a cornerstone in FAW control in North and South America [11,14].

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Conclusion