Cross-resistance And Molecular Mechanism Of Cry1ab-selected Ostrinia Furnacalis To Other Bt Toxins

Despite the proven track record of biotech Bt maize to provide an new tool for easier and more consistent Ostrinia furnacalis control, it is known for their ability to develop resistance in target pest to Bt toxins, particularly when the same or similar Bt maize expressing single toxin protein is planed in area wide. It is necessary to discover novel Bt genes, to develop novel insect resistant Bt maize with double or multiple genes, as well as to understand the molecular mechanism for evolution of resistance to Bt toxins in target pest, which will play an important roll in sustainable use of Bt maize as a valuable and innovative O. furnacalis control tactic through a management plan to delay or avoid the risk of resistance.Susceptibility to Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, and Cry1Ie proteins from Bt was determined for ACB-AbR and ACB-BtS strains of O. furnacalis, a Cry1Ab-selected strain and a Bt susceptible strain, through bioassay by exposing neonate larvae to semi-artificial diet incorporated with Bt proteins. Model Assays of competition binding to BBMV of O. furnacalis were performed with biotinylated Cry1Ac protein mixed with excessive unlabeled Cry1Ab, Cry1Ah, Cry1Fa, and Cry1Ie proteins, respectively. Binding receptors of Cry1Ac protein in BBMV of O. furnacalis were detected by using ligand blotting with biotainylated Cry1Ac protein. A proteomic approach was explored together with ligand blotting to identify Cry1Ab, Cry1Ac, Cry1Ah, and Cry1Ie binding proteins from ACB-BBMV. cDNAs of aminopeptidase N (APN) from both ACB-BtS and ACB-AbR strains were cloned and sequenced using RT-PCR and RACE technologies. The expressions of 4 APN genes and a known cadherin gene from O. furnacalis were detected by Real Time PCR.There were significant differences in susceptibility of ACB-BtS strain to Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, and Cry1Ie proteins. Their LC50 were 0.75, 2.37, 0.07, 2.19, and 1.87μg/g. ACB-AbR strain developed significan levels of resistance to Cry1Ab (39.7-fold), Cry1Ac (36.9-fold), Cry1Ah (131.7-fold), and Cry1Fa (6.2). In contrast, the susceptibility to Cry1Ie protein was not increased in ACB-AbR strain. This results susgest that the highest level of cross-resistance was observed with Cry1Ah (131.7-fold), followed by Cry1Ac (36.9-fold). A low level of cross-resistance (6.2-fold) to Cry1F was also detected. In contrast, ACB-AbR was equally susceptible to Cry1Ie as the unselected control strain.There were six binding peptides were identified as Cry1Ac binding proteins with the molecular weight 70, 120, 160, 200, 260, and 270kDa, respectively, among which the 120 and 200 kDa bindings were similar to that of APN and cadherin's molecular weights. The bindings of Cry1Ac protein to BBMV of O. funacalis were completely significantly inhibited by Cry1Ab and Cry1Ah proteins and partial inhibited by Cry1Fa protein. In contrast, no competitive binding was observed between Cry1Ie and Cry1Ac. These results indicated that Cry1Ac protein shares all receptors with Cry1Ab and/or Cry1Ah proteins in BBMV of O. furnacalis but partial with Cry1Fa protein. It was different in binding receptors between Cry1Ac and Cry1Ie proteins. Therefore, the variance of cross-resistance levels among 5 proteins was due to the difference in their bingding receptors on the BBMV. In addition, the V-type proton ATPase catalytic subunit A and heat shock 70 kDa proteins were identified as Cry toxins binding proteins in BBMV of O. furnacalis.Four APN isoforms, Ofapn1, Ofanp2, Ofapn3, and Ofapn4, were identified from O. furnacalis by cDNA cloning. The deduced amino acid sequences were included 994, 940, 1014, and 951 amino acid residues, respectively. The predicted molecular weights of Ofapn1, Ofapn2, Ofapn3 and Ofapn4 were 113.1, 106.8, 115.1, and 107.8kDa, respectively. Sequences analysis indicated that the deduced protein sequences are most similar to the aminopeptidases from Ostrinia nubilalis with 96%, 97%, 96%, and 96% sequence identity. Compared with APN sequences of ACB-BtS, there were 9, 5, 10, and 12 amino acid mutations in the deduced protein sequences of Ofapn1, Ofapn2, Ofapn3, and Ofapn4 in ACB-AbR, respectively. In addition, there was one more O-glycosylation site in Ofapn1r. At the non-translatable 3′-end region, a deletion of 11 nucleotides (positions 2828-2838) and an insert of 9 nucleotides (postion 2893-2901) occurred in the Ofapn2r. The expression of Ofapn1, Ofapn2, Ofapn3, and Ofapn4 mRNA were 2.03-fold, 1.61-fold, 2.69-fold, and 2.62-fold higher in ACB-AbR than in ACB-BtS. This suggest that the reduced susceptibility to Cry1Ab toxins in ACB-AbR strain may due to the increased ability of detoxification and repair effect of epithelial cell.