Identification of Cry1Ah-binding proteins through pull down and gene expression analysis in Cry1Ah-resistant and susceptible strains of Ostrinia furnacalis

Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis, Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling CrylAh to NHS-activated Sepharose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI, and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO.