Localization and functional studies of the cadherin BT-R1 as a Bacillus thuringiensis Cry1A-binding protein in the midgut of tobacco hornworm (Manduca sexta)
MetadataShow full item record
The members in Bacillus thuringiensis (Bt) Cry1A family have specific toxicity toward lepidopteran larva. A critical step in Bt-mediated insecticidal action is the binding of Bt toxin to its target molecules on insect midgut epithelium. Three Cry1A-binding proteins, aminopeptidase MsAPN1, the cadherin Bt-R1 and the membrane-type alkaline phosphatase (m-ALP), had been identified in the midgut of tobacco hornworm (Manduca sexta). Immunohistochemistry illustrated that these binding proteins had different localization on M. sexta larval midgut epithelia. To compare with the immunolocalization of Cry1A-binding proteins, Cry1A toxins showed co-localization on insect midgut microvilli. CR12-MPED, which is the membrane proximal part of Bt-R1, was identified as a critical region for Cry1Ab binding. This fragment was over-expressed and purified as a peptide from Escherichia coli. Unexpectedly, the Cry1Ab-induced M. sexta larval mortality was not blocked, but significantly enhanced by the addition of CR12-MPED. Further bioassays indicated that CR12-MPED could also potentiate Cry1Ac toxicity against tobacco budworm (Heliothis virescens) and corn earworm (Helicoverpa zea). When a putative Cry1A-binding epitope was removed from CR12-MPED, the derivative peptide (CR12-MPED/Del) lost binding to Cry1A 1toxins and the ability of toxicity enhancement. Far-UV circular dichroism (CD) and H-NMR spectroscopy showed that CR12-MPED was mainly composed of [beta]-strands and random coils in unfolded state. Histomicroscopy illustrated that CR12-MPED bound to midgut microvilli, but did not change Cry1A binding localization at these places. The CR12 sub-truncation was identified as the minimal region in CR12-MPED to enhance Cry1Ac toxicity against H. zea larvae. A proposed mechanism of this synergism was that the unfolded CR12-MPED accumulated onto microvilli through hydrophobic interactions, and Cry1A toxins could be attracted to these places by toxin-peptide binding, hence the Cry1A toxicity was increased. This discovery enables Cry1A toxins to effectively control lepidopteran pests even at low dosages, which can prevent insect resistance from high toxin selection pressure.