Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil

Highlights

• High level Cry1F resistance in fall armyworm from Brazil has been characterized at the molecular level.

• A full set of reference genes has been defined for future gene expression analysis in fall armyworm across larval stages.

• A GY deletion from the extracellular loop 4 of ABCC2 is linked with resistance to Cry1F.

• Functional expression of the wildtype and mutated ABCC2 gene in insect cells confirmed the significance of the GY deletion.

• Pooled population RNAseq and pyrosequencing of 40 field strains revealed that the GY deletion is fairly widespread.

Abstract

Fall armyworm, Spodoptera frugiperda (J.E. Smith) is a major lepidopteran pest of maize in Brazil and its control particularly relies on the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins such as Cry1F. However, control failures compromising the efficacy of this technology have been reported in many regions in Brazil, but the mechanism of Cry1F resistance in Brazilian fall armyworm populations remained elusive. Here we investigated the molecular mechanism of Cry1F resistance in two field-collected strains of S. frugiperda from Brazil exhibiting high levels of Cry1F resistance. We first rigorously evaluated several candidate reference genes for normalization of gene expression data across strains, larval instars and gut tissues, and identified ribosomal proteins L10, L17 and RPS3A to be most suitable. We then investigated the expression pattern of ten potential Bt toxin receptors/enzymes in both neonates and 2nd instar gut tissue of Cry1F resistant fall armyworm strains compared to a susceptible strain. Next we sequenced the ATP-dependent Binding Cassette subfamily C2 gene (ABCC2) and identified three mutated sites present in ABCC2 of both Cry1F resistant strains: two of them, a GY deletion (positions 788–789) and a P799 K/R amino acid substitution, located in a conserved region of ABCC2 extracellular loop 4 (EC4) and another amino acid substitution, G1088D, but in a less conserved region. We further characterized the role of the novel mutations present in EC4 by functionally expressing both wild type and mutated ABCC2 transporters in insect cell lines, and confirmed a critical role of both sites for Cry1F binding by cell viability assays. Finally, we assessed the frequency of the mutant alleles by pooled population sequencing and pyrosequencing in 40 fall armyworm populations collected from maize fields in different regions in Brazil. We found that the GY deletion being present at high frequency. However we also observed many rare alleles which disrupt residues between sites 783–799, and their diversity and abundance in field collected populations lends further support to the importance of the EC4 domain for Cry1F toxicity.

Introduction

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a polyphagous lepidopteran pest species, causing significant damage in several economically important crops, particularly maize in Brazil (Ivan Cruz, 1995; Barros et al., 2010). The control of this pest has relied mainly on synthetic insecticides, which led to the evolution of resistance to different chemical classes (Diez-RodríGuez and Omoto, 2001; Carvalho et al., 2013; Nascimento et al., 2016; Okuma et al., 2017; Bolzan et al., 2019). Currently, the main measure to control fall armyworm in Brazil involves the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins (O. Bernardi et al., 2015).

Since the introduction of transgenic maize expressing Cry1F in 2009, the Bt technology has been adopted in large scale year-round production in Brazil, with limited refuge areas of non-Bt plants (Horikoshi et al., 2016). Subsequently, the presence of the Cry1F protein in both maize and cotton products contributed to the evolution of Cry1F-resistance in fall armyworm, which was first reported in 2014 (Farias et al., 2014).

The inheritance of Cry1F resistance in Brazil has been described for S. frugiperda as (incompletely) recessive, autosomal and monogenic (Farias et al., 2014; Leite et al., 2016; Santos-Amaya et al., 2016a), and many recent studies have shown cross-resistance among Cry1F, Cry1A.105, Cry1Ac and Cry1Ab (Vélez et al., 2013; D. Bernardi et al., 2015; Santos-Amaya et al., 2016b; Burtet et al., 2017). Field-evolved resistance of fall armyworm to Cry1F in Argentina was characterized as autosomal and incompletely recessive (Chandrasena et al., 2018). In order to implement reliable resistance management strategies, it is important to understand the molecular mechanism of Bt resistance. A broadly accepted model of Bt toxicity is that once the crystalline inclusions containing the Cry proteins are ingested by the insect, they act in a sequential manner on different targets in the insect midgut. The Cry proteins have to be solubilized and processed to an active toxin. By crossing the peritrophic matrix, the activated toxins then interact with different enzymes and receptors, resulting in pore formation, osmotic cell lysis and insect death (Bravo et al., 2007; Adang et al., 2014). A number of proteins have been reported as receptors for the Cry toxins, including aminopeptidase N (APN), cadherin (CAD), alkaline phosphatases (ALP) and ATP-binding cassette (ABC) transporters (Bravo et al., 2007). A well-known Bt toxin resistance mechanism is the reduction of Cry toxin binding to their specific midgut receptors, by changes in the expression level and/or mutations (Bravo et al., 2007; Heckel et al., 2007). Many studies have indicated a major role of ABC transporter subfamily C2 (ABCC2) in mediating the insertion of Cry toxins into the midgut membrane of lepidopteran species (Gahan et al., 2010). Mutations in the ABCC2 transporters have been linked to Cry1-type resistance in many lepidopteran pests (Gahan et al., 2010; Atsumi et al., 2012; Park et al., 2014; Xiao et al., 2015), including S. frugiperda (Banerjee et al., 2017; Flagel et al., 2018). The resistance to Cry1F in fall armyworm populations from Puerto Rico has been linked to an insertion of two nucleotides in the ABCC2 gene, which lead to a premature stop codon and consequently a non-functional receptor for the Bt toxin (Banerjee et al., 2017; Flagel et al., 2018).

Nevertheless, until now the mechanism of Cry1F resistance in fall armyworm populations from Brazil is unknown. Therefore, understanding the molecular basis is critical to develop effective resistance management programs and sustain the Bt technology (Tabashnik et al., 2013). In the present study we elucidated the molecular mechanism of Cry1F resistance in S. frugiperda from Brazil. For this purpose we first selected stable reference genes with low expression variance among strains, larval stages and gut tissue. We then investigated in one susceptible and two Cry1F-resistant fall armyworm strains the expression pattern of known receptors/enzymes involved in Bt toxin mode of action. Next we screened for mutations in full length sequences of ABCC2 and characterized the functional role of non-synonomous mutations using cell toxicity assays. Finally, we assessed the frequency of the mutant alleles in fall armyworm populations recently collected from maize fields in different regions in Brazil by different technologies.