The soil bacterium Bacillus thuringiensis (Bt) is the most successfully used biopesticide in agriculture, and its insecticidal protein genes are the primary transgenes used for insect control in transgenic crops. However, evolution of insect resistance to Bt toxins threatens the long-term future of Bt applications. To date, cases of resistance to Bt toxins have been reported in agricultural situations in six insect species, but the molecular basis for these cases of resistance remains unclear. Here we report that the resistance to the Bt toxin Cry1Ac in the cabbage looper, Trichoplusia ni, evolved in greenhouses, is associated with differential alteration of two midgut aminopeptidases N, APN1 and APN6, conferred by a trans-regulatory mechanism. Biochemical, proteomic, and molecular analyses showed that in the Cry1Ac-resistant T. ni, APN1 was significantly down-regulated, whereas APN6 was significantly up-regulated. The Cry1Ac resistance was correlated with downregulation of APN1 but not with the up-regulation of APN6. The concurrent up-regulation of APN6 and down-regulation of APN1 might play a role in compensating for the loss of APN1 to minimize the fitness costs of the resistance. Along with identifying reduced expression of APN1 as the molecular basis of Bt resistance selected in an agricultural setting, our findings demonstrate the importance of APN1 to the mode of action of Bt toxin Cry1Ac.

No abstract

Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration.