An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis Cry1Ac Toxin

Abstract

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.

The Molecular Genetics of Insecticide Resistance

“…Thus, although ABC transporters have never been implicated in Bt resistance before, their association with this resistance locus strongly implicates this large class of receptors in Bt binding. Second, and perhaps most strikingly, similar putatively inactivating mutations are present in resistant strains of both H. virescens [a 22-bp frameshifting deletion (Gahan et al 2010)] and Plutella xylostella [a 30-bp deletion (Baxter et al 2011)]. This amazing finding suggests that parallel evolution in insecticide resistance is not confined to point mutations in ion channels but instead that even deletions in Bt targets can show remarkable evolutionary constraints.…”

“…For example, although numerous potential Cry1Ac-binding proteins have been identified from the brush border membrane of the lepidopteran midgut (Vadlamudi et al 1995;Nagamatsu et al 1998;McNall and Adang 2003), only one potential receptor, a 12-cadherin domain protein, has been linked to various resistant strains of the different caterpillar pests Heliothis virescens (Gahan et al 2001), Pectinophora gossypiella (Morin et al 2003), and Helicoverpa armigera (Xu et al 2005). This situation has now changed dramatically following the mapping of a second Cry1Ac resistance locus in H. virescens encoding the ABC transporter C2 (Gahan et al 2010). This finding is important for several reasons.…”

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

“…P-glycoproteins have been associated with resistance to cancer therapy drugs, conferring a multi-drug resistance phenotype when overproduced (Wang et al, 1995). ABC transporters have been shown to participate in insecticide resistance in various species, including mosquitoes (Porretta et al, 2008;Gahan et al, 2010;Bariami et al, 2012). ABC transporter mRNAs were also found overexpressed in Plutella xylostella strains resistant to chlorpyrifos (You et al, 2013).…”

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

“…Cry1AcMod was not more effective than Cry1Ac against two strains in which resistance was caused primarily by a mutant cadherin allele: the SCDr1 strain of H. armigera 18 and the YFO strain of H. virescens 24 (Fig. 2 and Supplementary Tables 4, 6 and 7).…”

“…24 We estimated resistance ratios for these strains based on the toxin concentration causing 50% larval growth inhibition (IC50). Relative to Cry1Ac, Cry1AcMod reduced the resistance ratio by a factor of >990 for YHD3 and ~100 for YFO (Supplementary Table 5).…”