MicroRNA-998–3p contributes to Cry1Ac-resistance by targeting ABCC2 in lepidopteran insects

Highlights

• MiR-998–3p was identified to target ABCC2 through a conserved binding site in diverse lepidopteran insects.

• MiR-998–3p could regulate the expression of ABCC2 in H. armigera, S. exigua and P. xylostella.

• Overexpression of miR-998–3p reduced Cry1Ac susceptibility in H. armigera, S. exigua and P. xylostella.

• MiR-998–3p may contribute to Cry1Ac-resistance by targeting ABCC2 in diverse lepidopteran insects.

Abstract

Cry protein toxins produced by Bacillus thuringiensis (Bt) are now widely used in sprays and transgenic crops to control insect pests. Most recently, ATP-binding cassette transporter proteins (ABC transporter), including ABCC2, ABCC3, ABCG1, ABCA2 and ABCB1, were reported as putative receptors for different Cry toxins. However, little is known about the regulatory mechanism involved in the expression of these ABC transporter genes. In the present study, a conserved target site of miR-998–3p was identified from the coding sequence (CDS) of ABCC2 in diverse lepidopteran insects. Luciferase reporter assays demonstrated that miR-998–3p could bind to the CDS of ABCC2 and down-regulate its expression through a conserved site and several non-conserved sites in three representative lepidopteran pests, including Helicoverpa armigera, Spodoptera exigua and Plutella xylostella. Injection of miR-998–3p agomir significantly reduced the abundance of ABCC2, accompanied by increased tolerance to Cry1Ac toxin in H. armigera, S. exigua and P. xylostella (Cry-S) larvae, while injection of miR-998–3p antagomir increased the abundance of ABCC2 dramatically, and thereby reduced the Cry1Ac resistance in a Cry1Ac resistant population of P. xylostella (GX-R). These results give a better understanding of the mechanisms of post-transcriptional regulation of ABCC2, and will be helpful for further studies on the role of miRNAs in the regulation of Cry1Ac resistance in lepidopteran pests.

Introduction

Bacillus thuringiensis (Bt) is an entomopathogenic gram-positive bacterium that produces insecticidal protein toxins (i.e., Cry, Cyt and Vip proteins) during sporulation, and has become the most extensively used microbial insecticide since the middle of last century (Schnepf et al., 1998; Sanahuja et al., 2011). These protein toxins are used in sprays and transgenic crops to control insect pests in field, but are safe to people or other non-target organisms (Mendelsohnet al., 2003; Sanchis, 2011; Liliana et al., 2013). In some cases, the application of Bt toxins reduced the use of chemical insecticides, increased crop yields, and consequently provided substantial environmental and economic benefits (Bravo et al., 2011). Unfortunately, resistance to Bt toxin has occurred in field populations of several major target insects, and threatened the efficacy of Bt toxin in the control of these insect pests (Badran et al., 2016; Tabashnik and Carrière, 2017). Thus, a comprehensive understanding of resistance mechanisms to Bt toxin is urgently required.

Cry protein toxins are the most commonly used Bt toxins in pest control. Cry toxins could be solubilized in the larval midgut and activated by gut proteases. The activated toxins then interact with multiple midgut membrane receptors, and result in perforation in the membrane of midgut epithelial cells and insect death (Martínez-Solís et al., 2018). Previous reports have indicated that specific toxin-receptor interaction was the key factor for toxicity, and many candidate receptors for Cry toxins have already been identified, such as classical cadherin (CAD) (Xie et al., 2005), ATP binding cassette transporter (ABC transporter) (Tanaka et al., 2013; Guo et al., 2015a; Stevens et al., 2017), alkaline phosphatase (ALP) (Jurat-Fuentes et al., 2011; Guo et al., 2015a), and aminopeptidase N (APN) (Sivakumar et al., 2007; Zhang et al., 2009).

Recently, ABC transporters are widely concerned due to their involvement in Bt resistance in various insects. Of which ABCC2 (Guo et al., 2015a; Stevens et al., 2017; Endo et al., 2018), ABCC3 (Guo et al., 2015a; Endo et al., 2018), ABCG1 (Guo et al., 2015b), ABCA2 (Tay et al., 2015) and ABCB1 (Zhou et al., 2019) have already been reported as Bt toxin receptors for different Cry toxins. Mutations of ABCC2 protein were considered as one of the main causes for Cry1Ac resistance in lepidopteran insects (Gahanet et al., 2010; Tanaka et al., 2016). Meanwhile, down-regulation of ABCC2 expression is also proved to be closely associated with Cry1Ac resistance. For example, Mitogen-activated protein kinase (MAPK) signaling pathway could alter the expression of midgut ALP, ABCC2 and ABCC3, and cause resistance to Cry1Ac toxin in Plutella xylostella (Guo et al., 2015a). Further, RNA interference (RNAi)-mediated knockdown (Guo et al., 2015a) or CRISPR/Cas9-mediated knockout (Guo et al., 2019) of ABCC2 in susceptible larvae could increase their resistance to Cry1Ac in P. xylostella. Most recently, a transcription factor, Forkhead box protein A (FOXA) has been shown to regulate the expression of ABCC2 and ABCC3, thereby affecting Cry1Ac-susceptibility in cell lines (Spodoptera litura Sl-HP cells and Spodoptera frugiperda Sf9 cells) and lepidopteran larvae (Helicoverpa armigera) (Li et al., 2017).

MicroRNAs (miRNAs) are small non-coding regulatory RNAs ranging from 19 to 24 nucleotides in length and act as important post-transcriptional regulators through targeting mRNA sequences, which may reside in the 5′untranslated region (5′UTR), the coding sequence (CDS) or the 3′ UTR of the target mRNA, and eventually cause cleavage, translational repression or mRNA decay (Behura, 2007; Asgari, 2013; Lucas et al., 2013). A large number of miRNAs have already been identified in a variety of insects (Liu et al., 2010; Liang et al., 2013; Mehrabadi et al., 2013; Etebari et al., 2013; Wang et al., 2016; Zhu et al., 2017), and some of them were confirmed to be involved in many important biological processes, such as insect metamorphosis (Ling et al., 2014; Yang et al., 2016; Song et al., 2018; Nouzova et al., 2018; Ye et al., 2019), reproduction (Ling et al., 2017; Song et al., 2019) and insecticide resistance (Hong et al., 2014; Li et al., 2015, 2019; Etebari et al., 2018). It should be pointed out that the functional identifications of miRNAs were often confined to a single insect species in the past. However, it is well known that miRNAs are often highly conserved across distantly related species (Ibanez-Ventoso et al., 2008), and the highly conserved miRNAs usually have the conserved target sites in specific target genes among different species (Bartel, 2009). For example, mRNA of ecdysone receptor (EcR) was confirmed as an important target of miR-14 in Drosophila melanogaster (Varghese and Cohen, 2007), H. armigera (Jayachandran et al., 2013), Bombyx mori (Liu et al., 2018) and Chilo suppressalis (He et al., 2019) from several independent studies. Thus, it is necessary to systematically explore the function of the conserved miRNAs among different insect species.

In the present research, a conserved target site of miR-998–3p was identified from the CDS of ABCC2 in diverse lepidopteran insects. Subsequently, miR-998–3p was proved to be able to regulate the expression of ABCC2, and thus mediate the Cry1Ac resistance in three representative lepidopteran pests, including H. armigera, Spodoptera exigua and P. xylostella. These results highlight the importance of miR-998–3p in the regulation of the expression of an important Cry1Ac receptor ABCC2 in lepidopteran pests.