Toxins · 14 authors, 5 centres
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This study used CRISPR/Cas9 to knock out the Pxpolycalin and PxABCC2 genes in Plutella xylostella (diamondback moth) and found that both mutations increased resistance to the Cry1Ac toxin. Genetic linkage analysis showed that PxABCC2, but not Pxpolycalin, was genetically linked to Cry1Ac resistance. The findings suggest PxABCC2 functions as a Bt receptor, while Pxpolycalin may contribute to resistance through a different mechanism.
**Background:** Bacillus thuringiensis (Bt) Cry toxins are widely used for pest control, but insects like Plutella xylostella have evolved resistance. Understanding the role of potential receptor proteins such as polycalin and ABCC2 in Bt resistance is critical for resistance management. Previous in vitro studies showed that polycalin binds Cry1Ac and Cry2Aa toxins, but its in vivo role in resistance remained unclear.
**Methods:** The study used the Cry1Ac-susceptible G88 strain and the Cry1Ac-resistant Cry1S1000 strain of P. xylostella. Full-length Pxpolycalin transcripts were sequenced from both strains. qRT-PCR was used to compare expression levels in midgut, across developmental stages, and in different tissues. Genetic linkage analysis was performed by crossing male Cry1S1000 with female G88, backcrossing F1 progeny with Cry1S1000, and genotyping backcross families with and without Cry1Ac selection (0.5 μg/mL). CRISPR/Cas9 was used to generate homozygous knockout strains: PoKO21KI16 (Pxpolycalin mutant with 16 bp insertion and 21 bp deletion in exon 2) and A3KO28 (PxABCC2 mutant). Bioassays were conducted at 0.50 and 0.25 μg/mL Cry1Ac toxin with five replicates.
**Key Results:** Sequencing revealed three fixed amino acid variations between susceptible and resistant strains (R/L1255L, R/Q2097Q, A/T2942T), with the second mutation located on a ligand binding site. qRT-PCR showed significantly higher Pxpolycalin expression in the G88 susceptible strain midgut compared to Cry1S1000 (p < 0.05). Expression was highest in third instar larvae (susceptible) and second instar (resistant), and was predominantly in midgut tissue. After Cry1Ac treatment (LC10, 0.10 μg/mL), both PxABCC2 and Pxpolycalin transcript levels increased from 6 to 36 h, peaking at 36 h, with a transient initial reduction. Genetic linkage analysis showed that Pxpolycalin gene mutations and transcript levels were not linked to Cry1Ac resistance — resistant-allele (RPoRPo) and heterozygous (RPoSPo) genotypes segregated near 1:1 in backcross families regardless of Cry1Ac selection. In contrast, PxABCC2 showed tight genetic linkage: backcross family a had a 14:10 ratio (Fisher's exact test, χ² = 11.46, df = 1, p < 0.001) and family b had 15:9 (χ² = 12.86, df = 1, p < 0.000) for R A2 R A2 :R A2 S A2 genotypes. Expression level linkage analysis confirmed PxABCC2 co-segregation with resistance (8:12 separation ratio, χ² = 10.00, df = 1, p < 0.05). Bioassays showed that at 0.50 μg/mL Cry1Ac, mortality of A3KO28 (14.00 ± 2.00%) was significantly lower than PoKO21KI16 (77.55 ± 5.00%), and both were significantly lower than wild-type G88 (one-way ANOVA, Tukey's test, p < 0.05). Compared to G88, mortality decreased by 1.29-fold for PoKO21KI16 and 7.14-fold for A3KO28.
**Clinical Implications:** This study provides in vivo evidence that PxABCC2 functions as a Bt Cry1Ac receptor and is genetically linked to resistance in P. xylostella, while Pxpolycalin, despite binding Bt toxins in vitro, is not genetically linked to resistance and likely contributes through an alternative pathway. These findings refine the understanding of Bt resistance mechanisms and inform resistance management strategies for diamondback moth, a major agricultural pest. The lack of synergistic effect in double mutants (unpublished data) further supports that polycalin does not act as a classical Bt receptor.