The role of GPI-anchored membrane-bound alkaline phosphatase in the mode of action of Bt Cry1A toxins in the diamondback moth

The insecticidal Cry proteins produced by the bacterium Bacillus thuringiensis (Bt) are extensively used for pest control in formulated sprays and in genetically modified crops, but resistance to Bt toxins threatens their sustainable use in agriculture. Understanding the molecular mechanisms involved in Bt pathogenesis is crucial for the development of effective resistance management strategies. Previously, we showed a strong correlation between Cry1Ac resistance in Plutella xylostella (L.) and down-regulation of the glycosylphosphatidylinositol (GPI)-anchored membrane-bound alkaline phosphatase (mALP) and aminopeptidase (APN) and members of the ATP-binding cassette (ABC) transporter subfamily C (ABCC), but we do not yet have a clear understanding of the relative contribution of each midgut receptor type. Here, a P. xylostella strain homozygous for the PxmALP gene knockout was generated using CRISPR/Cas9 and the results showed that this strain had a 294-fold resistance to Cry1Ac toxin and 394-fold cross-resistance to Cry1Ab. Moreover, a triple knockout strain lacking PxmALP, PxABCC2, and PxABCC3 exhibited 9,660-fold resistance to Cry1Ac and 5,662-fold cross-resistance to Cry1Ab. These resistance levels surpassed those observed in the previously described double PxABCC2 and PxABCC3 knockout mutant, revealing a functional redundancy between ABC transporters and PxmALP. In addition, the activity of Cry1A toxins against Sf9 cells expressing PxmALP, PxABCC2 or PxABCC3 confirmed that each of these can act as a functional receptor. Our findings are crucial for unraveling the relative role of multiple receptors and the molecular mechanisms underlying Bt resistance in insects.