The cDNA encoding 120-kDa aminopeptidase (APN) of Bombyx mori was expressed transiently in cultured Drosophila (DM-S2) cells using a heat shock promoter. APN was expressed primarily on the surface of DM-S2 cells. Based on immunostaining about 15 to 20% of the cells expressed APN. Bacillus thuringiensis Cry1Aa toxin bound only to the surface of cells that expressed B. mori APN and no Cry1Aa binding was detected to DM-S2 cells expressing Manduca sexta 120-kDa APN. After treatment with Cry1Aa, about 16% of the cells transfected with pHSP-BmAPN were dead, while 8% of the cells transfected with control pHSP-HR5 were killed. When cells transfected with pHSP-BmAPN and pHSP-HR5 were treated with Cry1Ac, no significant difference between treatments was measured. To confirm the relationship between APN expression and Cry1Aa toxicity, we co-expressed APN under the control of opIE-2 promoter with green fluorescent protein marker using plasmid pIZT/V5-His. Cry1Aa toxicity to cells was visualized using ethidium homodimer-1. After treatment with Cry1Aa, cells that co-expressed GFP and APN were killed. In this study we co-localized B. mori APN expression with Cry1Aa binding and toxicity, thereby demonstrating the function of this APN as a Cry1Aa receptor.
Species 1: Bombyx mori (silk moth)
Species 2: Manduca sexta (tobacco hornworm)
Keywords: Bacillus thuringiensis, aminopeptidase
The ESA 2001 Annual Meeting - 2001: An Entomological Odyssey of ESA