Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

Abstract

ABSTRACTAminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium ofManduca sextahave been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced byBacillus thuringiensissubsp.kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles ofM. sextashowed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned fromM. sextamidgut RNA and expressed inEscherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting thatB. thuringiensissubsp.kurstakiproduces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.