Molecular Mechanism Of Receptor-mediated Differential Sensitivities Of Three Different Spodoptera (Noctuidae) Species To Bacillus Thuringiensis Toxin

Bacillus thuringiensis is a widely used microbial pesticide in the world,The Cry toxin produced by Bacillus thuringiensis could directly act on pests.Transfering the Bt genes into crops could reduce the application amount of chemical pesticides.Bt biological pesticide has been widely used in agricultural pest control for a long time beacause of its harmless human beings and friendly to environment.The three Spodoptera(Noctuidae)species S.litura,S.exigua and S.frugiperda are different kinds of omnivorous insects with strong migratory ability which are harmful to agricultural crops all over the world.The difference in sensitivity of these three insects to Bt toxin was detected,And we investigated thoroughly at the molecular level to understand the mechanisms that lead to these differences.It provides theoretical support for effective prevention and control of agricultural pests.1 Analysis of the sensitivity of Bt toxin to three Spodoptera(Noctuidae)species.The susceptibility of different Lepidopteran insects to Bt toxin was different.There are two causes,including the differences in sensitivity mediated by toxin activation processes or the binding of toxin to receptor proteins.The sensitivity of Cry toxin to three kinds of insects of Spodoptera(Noctuidae)insects was tested.The results showed that the survival rates were 96.43%,72.73%and 0 respectively after 7 days treatment with the 2nd instar S.litura,S.exigua and S.frugiperda by 40 ?g/g Cry1Ac toxin.And the sensitivity of the three insects to Cry 1 Ac toxin was significantly different(p<0.05).The survival rates were 48.21%,47.27%and 3.63%respectively after 7 days treatment with the three 2nd instar Spodoptera(Noctuidae)species by 40 ?g/g Cry1Ca toxin.There was no difference in the sensitivity to Cry1Ca toxin between S.litura and S.exigua,but the sensitivity of these two insects to Cry1Ca was significantly lower than that of S.frugiperda that to Cry1Ca toxin(p<0.05).Based on weight change and survival analysis,sensitivity of S.litura and S.exigua to Cry1Ca toxin were stronger than to Cry1Ac(p<0.05).However the S.frugiperda showed was relative sensitive to both Cry 1 Ac and Cry1Ca toxins.2 Cloning and bioinformatics analysis of ABCC2 transporter and Cadherin genesIt was the main reason for the differential susceptibility of insects to Bt toxin that the differences in sensitivity mediated by binding of toxin to receptor proteins.ABCC2 transporter and cadherin were two important toxin receptor proteins in the process of action of Bt toxin on Lepidopteran insects.We have constructed three expression plasmids pie2-S1ABCC2-GFP,pie2-SeABCC2-GFP and pie2-SfABCC2-GFP whose genes were from the midgut of the three Spodoptera(Noctuidae)insect larvae or its cell line.We found all the three expressed proteins are mainly located in the cell membrane.SlABCC2,SeABCC2 and SfABCC2 genes could encode 1344,1343 and 1345 amino acid residues and their molecular weights were 150.15 kDa,149.74 kDa and 150.25 kDa,respectively.And the three ABCC2 proteins were highly conserved.Amino acid sequence analysis revealed that they share 97.42%identity among all the three ABCC2 proteins.They have 12 transmembrane structures domains.We have constructed the two expression plasmids pie2-SlCAD-GFP,pie2-SeCAD-GFP whose genes were from the midgut of S.litura and S.exigua.And we constructed expression plasmid pie2-SfCAD-GFP using SfCAD gene synthesized by company.The observation of fluorescence microscope revealed that pie2-SlCAD-GFP,pie2-SeCAD-GFP and pie2-SfCAD-GFP were mainly located in the cell membrane.SlCAD,SeCAD and SfCAD genes could code for 1757,1739 and 1734 amino acid residues and their molecular weight were 197.47 kDa,196.45 kDa and 195.50 kDa,respectively.Amino acid sequence analysis revealed that shared 83.92%identity among the three cadherin proteins3 Synergistic effects between ABCC2 transporter and cadherinIn order to investigate whether ABCC2 transporter and cadherin could act as specific receptors of Cry toxin in the three Spodoptera(Noctuidae)insects,The constructed expression plasmids were transfected into Hi5 cells.The EC50 of activated Cry1Ac toxin to Hi5 cells with expressing SlABCC2,SeABCC2 and SfABCC2 were 4.198 ?g/mL,0.092?g/mL and 0.064 ?g/mL,respectively.Conversely,Cry1 Ac toxin was no toxicity to Hi5 cells expressing SlCAD,SeCAD and SfCAD.Therefore,it seems that three cadherins of Spodoptera(Noctuidae)insects were not Cry 1 Ac toxin receptors at the cellular level.We treated the Hi5 cells transfected with the 6 plasmids using Cry1Ca toxin,respectively.The results showed there were no significant difference between treatment groups and control.It seems neither ABCC2 nor CAD of three Spodoptera(Noctuidae)insects could act as Cry1Ca toxin receptors at the cellular level.The susceptibility of Hi5 cells co-transfected with pie2-HaABCC2-GFP and pie2-HaCAD-GFP to Cry1Ac was 6.41-fold and 541.88-fold more than that of Hi5 cells transfected with pie2-HaABCC2-GFP and pie2-HaCAD-GFP individually.It showed that combination of HaABCC2 with HaCAD could enhance the sensitivity of Hi5 cells to Cry 1 Ac toxin.In the other words,Hi5 cells co-transfected with Helicoverpa armigera HaABCC2-GFP and pie2-HaCAD-GFP showed strong synergistic effects to Cry 1 Ac toxin.Then we co-expressed of ABCC2 and CAD of all the three Spodoptera(Noctuidae)insects in Hi5 cells,respectively.The results demonstrated that the co-expression in three groups could not enhance the sensitivity of Hi5 cells to Cry1Ac toxin or Cry1Ca toxin.Thus,there were no synergistic effects to Cry 1 Ac toxin and Cry1Ca between ABCC2 and cadherin in all the three Spodoptera(Noctuidae)insects,respectively.4 Molecular mechanism of differential sensitivities to Cry1Ac among three Spodoptera(Noctuidae)speciesBacillus thuringiensis Cry toxins exert their toxicity by forming membrane pores after binding with larval midgut membrane proteins known as receptors.S.litura and S.frugiperda belong to the same genus,but S.litura is tolerant to Cry1Ac,while S.frugiperda is relative susceptible.The mechanism involved in the differential toxicity of Cry1Ac to these insect species is not understood.Amino acid sequences analysis of ABCC2,a well-recognized Cry1Ac receptor,from both species showed high sequence identity.Hi5 insect cells expressing SfABCC2 from S.frugiperda cell line were 65.59-fold more susceptible than those expressing the SlABCC2 from S.litura larvae.Substitution of fragments,point mutations and deletions between the ABCC2 of the two species revealed that ABCC2 amino acid Q125 from SfABCC2 or E125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors.Consistently with this,cells expressing Helicoverpa armigera HaABCC2Q122-GFP,were more susceptible to Cry1Ac than cells expressing HaABCC2E122-GFP mutant.Q125 or E125 is located in a predicted exposed loop 1 region of ABCC2 indicating that this region could be important for Cry1Ac binding.These findings identified a single amino acid residue located in loop 1 of ABCC2 transporter as responsible for the different levels of susceptibility to Cry1Ac among various lepidopteran species.