Genome mapping coupled with CRISPR gene editing reveals a P450 gene confers avermectin resistance in the beet armyworm

Abstract

The evo of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm, Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance.

Functionally characterized arthropod pest and pollinator cytochrome P450s associated with xenobiotic metabolism

“…The validation systems used for each P450 are mentioned in Table 2 (last two columns). It is notable that CRISPR in non-model organism has been so far utilized only for Lepidoptera P450 validation (H. armigera 6AE cluster, and S. exigua 9A186 (Zuo et al, 2021)). (a) Twenty two P450s (Figure 3B) have been validated to resist/tolerate xenobiotics using only the system of RNAi in Lepidoptera, despite the debate in the literature concerning the effectiveness of RNAi in this order (Terenius et al, 2011).…”

“…For example, all the P450s from Spodoptera litura (CYP321B1, CYP6B50, CYP6AB14, CYP9A40, CYP6AB60, CYP6AB12) have been validated for their contribution to resistance using RNAi (Lu et al, 2020(Lu et al, , 2019b(Lu et al, , 2019aSun et al, 2019;Wang et al, 2015bWang et al, , 2015a. Eighteen Lepidoptera P450s have been validated using in vitro systems only, like H. zea CYP6B27 (Wen et al, 2009) and H. armigera CYP9A12 (Chen et al, 2019;Shi et al, 2021;Tian et al, 2019;Yang et al, 2008), while only one P450 (H. armigera CYP6AE20) has been validated using only genome modification approaches (Figure 3B and Table 2A). Additionally, two P450s have been verified using both RNAi and in vitro systems: H. armigera CYP6B6 and CYP9A14 (Shi et al, 2021;Tao et al, 2012;Tian et al, 2017;Yang et al, 2008;Zhao et al, 2016) while 11 P450s have been verified using both in vitro and genome modification systems: the H. armigera 6AE cluster CYP6AE11, 6AE12, 6AE15, 6AE16, 6AE17, 6AE18, 6AE19 (both heterologous expression and CRISPR) and S. exigua CYP321A16, CYP332A1, CYP321A8 (both heterologous expression and D. melanogaster transgenic expression) (Bo et al, 2020) and CYP9A168 both heterologous expression and D.…”

“…melanogaster system (Zuo et al, 2021) (Figure 3Band Table 2A). H. armigera CYP6AE14 has been validated for xenobiotic tolerance and metabolism using all three categories: in vitro systems, genome modification and RNAi (Mao et al, 2007;Tao et al, 2012)(Figure 3B and Table 2A);…”

“…In vivo functional validation of the role of P450s to xenobiotic tolerance and resistance has been facilitated by the RNA interference (RNAi) approach (Bai-Zhong et al, 2020;Ding et al, 2013;Gao et al, 2016;Kalsi and Palli, 2017;Mao et al, 2007;Pang et al, 2016; as well as Drosophila transformation tools (Daborn et al, 2012;Manjon et al, 2018;Riga et al, 2020;Samantsidis et al, 2020;Troczka et al, 2019;Tsakireli et al, 2019;Zhu et al, 2010;Zimmer et al, 2018)and the recent advances in genome editing technology in non-model organisms Zuo et al, 2021).…”

Transcriptomic and proteomic analysis of pyrethroid resistance in the CKR strain of Aedes aegypti

“…In order to understand the molecular basis of CYP-mediated resistance we considered six testable hypotheses (Table 1). One hypothesis (#1) was that resistance was due to a polymorphism in a CYP resulting in an enzyme with a higher rate of detoxification of pyrethroids (this was previously shown for Cyp6a2 and metabolism of DDT [40] and for CYP9A186 and abamectin metabolism [41]). Two hypotheses involved increased expression of one CYP due to a mutation in the CYP "promoter" (hypothesis #2, as found for CYP6D1 and metabolism of pyrethroids, [42][43][44]) or due to gene duplication (hypothesis #3, as found for resistance to neonicotinoids due to duplication of CYP6CY3 [45]).…”