In vitro hydrolysis of Bacillus thuringiensis Cry1Ac toxin by gut proteases of Nilaparvata lugens (Stål) and binding assays of Cry1Ac toxin with brush border membrane of N. lugens midgut

Bacillus thuringiensis(Bt) and transgenic crops carryingcrygenes arewidely used in the management of lepidopteran and coleopteranpests. However, almost none of the Cry toxins have insecticidalproperties against sap-sucking insects, such as planthoppers,leafhoppers and aphids. To understand the low insecticidalactivity of Cry1Ac toxin on sap-sucking insects, we investigatedtwo critical steps in the Bt-intoxication cascade: the proteolyticprocessing of Cry1Ac toxin by gut proteases, and the binding ofCry1Ac to brush border membrane vesicles (BBMV) ofNilaparvatalugens. Proteolytic processing of Cry1Ac protoxin byN. lugensgutproteases resulted in an∼65 kDa product, similar to the expectedsize of the trypsin-activated Cry1Ac toxin. In addition, activation ofcysteine proteases inN. lugensgut increased the efficiency ofproteolytic activities in the processing of Cry1Ac. However,feedingN. lugensnymphs with either Cry1Ac protoxin or trypsin-activated Cry1Ac toxin resulted in low mortalities. The LC50ofCry1Ac protoxin and trypsin-activated Cry1Ac was 198.92 and450.18μg/mL, respectively.In vitrobinding analysis of BBMV withthe pre-activated Cry1Ac showed that Cry1Ac toxin couldspecifically bind to the BBMV. However, binding competition with500-fold molar excess GalNAc (N-acetyl-D-galactosamine)suggested that the binding was not mediated by GalNAc-likeglycoproteins. These results indicate that Cry1Ac toxin could besuccessfully processed by the treatment ofN. lugensgutproteases. However, the binding of Cry1Ac toxin to the midgutbrush border membrane was not mediated by GalNAc-likeglycoprotein. This may be responsible for the low susceptibility ofN. lugensto Cry1Ac