D3247: Functional examination of aminopeptidase N1 in resistance to Bt toxins in Trichoplusia ni

The membrane-bound aminopeptidase N1 (APN1) in insect midgut is one of the proposed receptors for Bacillus thuringiensis toxins. In the cabbage looper, Trichoplusia ni, the high-level resistance to Bt toxin Cry1Ac has been identified to be associated with both a frame-shift mutation in the CDS of the ABC transporter ABCC2 gene and the lack of 110 kDa APN1 protein in the midgut brush border membrane. However, the functional role of APN1 in the pathway of Bt toxicity requires to be understood. In this study, the expression of APN1 gene in the larval midgut of Cry1A-resistant T. ni and the Cry1Ac-resistant diamondback moth Plutella xylostella was analyzed and significant down-regulation of the gene was determined. To functionally examine the effect of lack of APN1 in larval midgut on response to Bt toxins in T. ni, four APN1 mutant T. ni strains were generated by CRISPR/Cas9 mutagenesis. Protein analysis confirmed that the APN1 in the midgut brush border membrane was not affected by the ABCC2 gene and the APN1 protein was completely absent in the four APN1 mutant strains generated in this study. Bioassays of the mutant T. ni strain, in comparison with the wildtype strain, with Cry1 toxins showed that knockout of APN1 alone did not confer significant resistance to the Bt toxins, and how the observed lack of APN1 together with ABCC2 gene mutation confers high-level resistance requires further understanding.