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Title: Identification of a novel aminopeptidase p-like gene (OnAPP) and it's possible involvement in Bt toxicity and resistance in a major corn pest(Ostrinia nubilalis)

Author
item KHAJURIA, CHITVAN - Kansas State University
item BUSCHMAN, LARRY - Kansas State University
item Chen, Ming-Shun
item SIEGFRIED, BLAIR - University Of Nebraska
item ZHU, KUN YAN - Kansas State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/1/2011
Publication Date: 8/24/2011
Citation: Khajuria, C., Buschman, L.L., Chen, M., Siegfried, B.D., Zhu, K. 2011. Identification of a novel aminopeptidase p-like gene (OnAPP) and it's possible involvement in Bt toxicity and resistance in a major corn pest(Ostrinia nubilalis). PLoS One. 6(8):e23983.

Interpretive Summary: The use of Bacillus thuringiensis (Bt) toxins in transgenic plants has revolutionized insect pest management in crops including corn, cotton, and other crops. However, there is a potential danger for the Bt strategy since insects can develop resistance to Bt toxins. To maximize and prolong the effectiveness of Bt toxins, we need to understand the mechanisms for insects to develop resistance to these toxins. This research characterized 10 aminopeptidase-like genes in the insect pest European corn borer (ECB), and analyzed their potential involvement in insect resistance to the Bt Cry1Ab toxin. We found that the two amino acids of an aminopeptidase-P like protein, Glu305 and Arg307, were changed to Lys305 and Leu307 in two Cry1Ab-resistant ECB strains compared with three Cry1Ab-susceptible strains. Suppression of the aminopeptidase-P-like transcript level by RNA-interference resulted in ECB larvae that were more resistant to the Bt toxin Cry1Ab. These results suggest that the aminopeptidase-like gene may be involved in conferring Bt toxicity and resistance in ECB resistance strains.

Technical Abstract: Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study we identified and characterized 10 aminopeptidase-like genes in relation to Bt Cry1Ab toxicity and resistance. The expression analysis for 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut. No difference was found in the expression of these genes between Cry1Ab resistant and susceptible strains, which suggest that altered expression of these genes is unlikely to be responsible for resistance in these ECB strains. However, we found changes in two amino acids of the amino acid sequence of aminopeptidase-P like gene (OnAPP), Glu305 changed to Lys305 and Arg307 changed to Leu307 in two Cry1Ab resistant strains as compared with three Cry1Ab-susceptible strains. OnAPP is 701 amino acids long and has putative signal peptide at N-terminal, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. Using feeding based-RNA interference assay, we achieved 38 % reduction in the level of OnAPP transcript as compared with the control after 8 days. Furthermore, a Bt bioassay using insects fed diet containing OnAPP dsRNA resulted in reduced susceptibility to Cry1Ab by 25% as compared with larvae fed GFP dsRNA. These results strongly suggest that OnAPP gene may be involved in conferring Bt toxicity and resistance in two ECB strains.