Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Larvae to subsp. Cry4Ba and Cry11Aa Toxins.

The insecticidal Cry4Ba and Cry11Aa crystal proteins from subsp. (Bti) are highly toxic to larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of to Bti toxicity . In this study, two Cry4Ba/Cry11Aa-binding GPI-APN isoforms (APN1 and APN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the APN1/APN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of APN1 or APN2, nor the simultaneous disruption of APN1 and APN2 resulted in significant changes in susceptibility of larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that APN1 and APN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in larvae as their binding proteins.