Abstract
The diamondback moth, Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacterium Bacillus thuringiensis (Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal that P. xylostella requires combined mutations in both PxABCC2 and PxABCC3 to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations of PxABCC2 and PxABCC3 that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella, with a mutation (RA2) causing the mis-splicing of PxABCC2 and another mutation (RA3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed that RA2 and RA3 were tightly linked to Cry1Ac resistance. Introgression of RA2 and RA3 enabled a susceptible strain (G88) of P. xylostella to obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role of PxABCC2 and PxABCC3 in Cry1Ac resistance, frameshift mutations were introduced into PxABCC2 and PxABCC3 singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion of PxABCC2 or PxABCC3 alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance in P. xylostella by demonstrating mutations within both PxABCC2 and PxABCC3 genes are required for high-level Cry1Ac resistance.
Highlights
Bacillus thuringiensis (Bt) is a ubiquitous gram-positive bacterium that can produce insecticidal protein toxins used for the control of many lepidopteran, coleopteran and dipteran pests [1,2]
Multiple studies have reported that ATP-binding cassette (ABC) transporters are important Bt receptors, and mutations in either ABC transporter subfamily C2 (ABCC2) or ABC transporter subfamily C3 (ABCC3) can lead to Cry1Ac-toxin resistance, this process is not fully understood
We identified inactivating mutations in these two genes from a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella and conducted genetic linkage analysis, which supported the role that PxABCC2 and PxABCC3 were the causal genes of Cry1Ac resistance
Summary
Bacillus thuringiensis (Bt) is a ubiquitous gram-positive bacterium that can produce insecticidal protein toxins used for the control of many lepidopteran, coleopteran and dipteran pests [1,2]. Mutations in ABC transporter subfamily C2 (ABCC2) are associated with Bt (Cry1A toxins) resistance in a number of lepidopteran species [11,12,13,14,15,16,17]. The receptor functionality of ABCC2 for Cry1A toxins has been well characterized using heterologous expression in cultured cell lines, manipulating mRNA abundance with RNA interference and with transgenic Drosophila [14,18,19,20,21,22]. ABC transporter subfamily C3 (ABCC3), a paralog of ABCC2, has been reported to mediate Cry1A toxicity [14,22,23,24]. Wang et al [25] reported mutations in ABCC2 plus ABCC3 were required to achieve highlevel Cry1Ac resistance in Helicoverpa armigera, suggesting functional redundancy as toxin receptors
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