Mechanism Of Resistance To Bt Toxin In Insect Cells Via Mutation Of Cadherin

Bacillus thuringiens(Bt)produces insecticidal crystal proteins that can safely and effectively kill agricultural and sanitary pests.Helicoverpa armigera is a multi-food agricultural pest that causes serious damage to a wide range of crops such as cotton,which causes significant economic losses to the global agriculture each year.Bt transgenic cotton is widely used in the world to control pests such as cotton bollworm.However,long-term large-scale use of Bt toxins leads to insect resistance to Bt toxins,which greatly reduces the effective control of Bt transgenic crops and Bt products.Some of the resistance mechanism are described such as early translation-termination,reduced gene-expression of Cry toxins receptors orreduced binding of Cry toxins to receptors.Some mechanisms are not completely elucidated.Previous study found that Cry1Ac resistance of Helicoverpa armigera 96CADwas tightly linked to the cadherin allele compared to susceptible Helicoverpa armigera 96S.By amino acid sequence alignment,it was found that there were 35 different amino acid residues between cadherin protein(mHaCAD)and 96S(HaCAD)cadherin in 96CAD.In vitro experiments showed that the binding of Cry1Ac toxin to cadherin in susceptible and resistant Helicoverpa armigera was similar,and the expression of cadherin in the two Helicoverpa armigera was not significantly different at mRNA level.But the resistance mechanism has not been definited.This study found that the distribution of mHaCAD protein in the 96CAD midgut epithelial cell brush border membrane(BBM)was significantly lower than the 96S strain.Expression of both cadherin alleles from 96CAD and 96S in Hi5 and immunofluorescence showed that the 96S-HaCAD protein was mainly localized on the cell membrane and the 96CAD-mHaCAD protein was mainly retained on the endoplasmic reticulum.By means of fragment replacement and immunofluorescence techniques,it was found that the mechanism of the differential localization of cadherin in the resistant Helicoverpa armigera was the mutation of the 172nd amino acid(D172G).These results demonstrated that changes of the localization of cadherin in cells may lead to cell resistance to Cry1Ac toxin.Although the lack of cadherin and other receptor proteins in the microvilli of the midgut epithelium of insects is a well recognized mechanism of resistance to Bt toxins.However,this study has found that mutations affecting membrane receptor trafficking can lead to a reduction in receptors on the cell membrane,and this is a new mechanism that also results in resistance to Bt toxins,which has not previously been reported.In addition,it was also found that the location of some mutants-expressed in Hi5 had no significant changes after the deletion of the different domains of susceptible Helicoverpa armigera,If the proximal extracellular domain,toxin binding region,or partial calcium ion binding region is deleted,the susceptibility to Cry 1 Ac toxin of these transient-expressing mutants was significantly reduced.This suggests that a variety of mutations in the protein of cadherin can cause insects to produce high-level resistance to Cry1Ac toxins.