Pesticidal and Receptor Binding Properties of Bacillus thuringiensis Cry1Ab and Cry1Ac δ-Endotoxin Mutants to Pectinophora gossypiella and Helicoverpa zea

Abstract

Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's (³⁶⁸RR³⁶⁹) of Cry1Ab and Cry1Ac toxins were replaced with Ala (³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, ³⁶⁸AA³⁶⁹), Glu (³⁶⁸EE³⁶⁹), Phe (³⁶⁸FF³⁶⁹), His (³⁶⁸HH³⁶⁹), and Lys (³⁶⁸KK³⁶⁹). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding ³⁶⁸AR³⁶⁹, produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, ³⁶⁸AR³⁶⁹, ³⁶⁸AA³⁶⁹, ³⁶⁸FF³⁶⁹, and ³⁶⁸HH³⁶⁹, lost toxicity; ³⁶⁸EE³⁶⁹ had reduced toxicity; whereas the more conserved change ³⁶⁸KK³⁶⁹ retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, ³⁶⁸AA³⁶⁹ and ³⁶⁸FF³⁶⁹, abolished the toxicity. Double mutant ³⁶⁸KK³⁶⁹ of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants ³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, and ³⁶⁸HH³⁶⁹ retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except ³⁶⁸KK³⁶⁹. In Cry1Ac single mutants, ³⁶⁸AR³⁶⁹ and ³⁶⁸RA³⁶⁹, reduction in the toxicity was observed. A double mutant of Cry1Ac, ³⁶⁸KK³⁶⁹, also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of I_sc across the transepithelial membrane of the insect midgut.