Identification Of Putative Receptors And Screening Of Mimics For Two Cry Toxins In Ostrinia Furnacalis(Guenee)and Plutella Xylostella

Bacillus thuringiensis(Bt)is the most widely used biological insecticide with great development space,which can form the poisonous parasporal crystal toxins(Cry)during spore formation,The Cry toxins can cause the lysis of cells,resulting in growth inhibition or death of target insects.Accompanied by the development of Gene-modified(GM)technology,the cry genes have made significant positive effects on the prevention against Ostrinia furnacalis,Helicoverpa armigera and Plutella xylostella,and drove the formation of regulations for the GM crops(GMCs),and bring enormous economic efficiency in agricultural production.However,the widespread adoption of Bt GMCs may increase the potential risk of resistance in pests,which threatens the effects of prevention and control.In addition,the problems of ecological impacts on the soil system,gene flow,putative immunotoxicity in mammals caused by the cultivation of Bt crops and the security of GM technology have also attracted wide attention.The development of insecticidal proteins with security and high virulence,and the modification of cry genes to enhance the toxicity and safety of existing Cry toxins have always been an important direction of pesticide research.Currently,the reports on the modification of active structures of existing Cry toxins based on the resistance genes of pests are most prevalent.Previous studies have shown that there are high similarities between heavy chain variable region(VH)and Domain ? or ? of Cry toxins in three dimensional(3D)space structure,and VHs have the potentials to mimic Cry toxins.This finding could provide an important reference for improving the biological activities of Cry toxins.With the invasion and harm of Cry IF toxin target pest Spodoptera frugiperda in China,as well as the development of resistance management,risk assessment and other technologies,the promotion of Cry1F-maize and other Cry1F-GMCs might be expected in China.However,there are few reports on the putative receptors and mechanisms of Cry 1F toxin in O.furnacalis(Guenee),which is one of the target pests of Cry IF toxin could cause severe yield reductions of maize and other crop in our country.The objective of this work is focusing on developing the mimics of Cry toxins,simulating the Cry1Ac and the Cry1F toxins combined with the phage antibody library technology,screening targeted with receptor and anti-idiotypic antibody,and evaluating the biological activities of mimics and splicing products.Moreover,the binding proteins of Cry1F toxin in the midgut of O.furnacalis(Guenée)were enriched and screened,and the functional verification for the putative receptors were performed by RNAi technology.This report endeavors to explore the high-efficiency technical route for the development of the innovative biological pesticides,and provides the references for progress on correlation between structure and activity of Cry toxin,and the interaction of the Cry toxin with its targeted receptors.1.Screening and bioactivity identification for the mimics of Cry1Ac toxin and its fusion proteinsBased on the previous report on the interaction between the Cry1Ac toxin-binding region of P.xyllostella cadherin-like receptor(PxCad TBR)with the Cry1Ac toxin,the region of PxCad TBR was expressed in E.coli BL21(DH3)and purified,which was used to screen targeted with human single-domain antibody phage display(Dab library).After three rounds of the panning process,three positive clones were identified which specifically bind to the region of PxCad TBR.Then the positive clone 4E12-1 was expressed in E.coli BL21(DH3)and purified through Ni-chelating affinity chromatography,and then the activities of binding,cytotoxicity and toxicity of 4E12-1 were preliminarily identified.The results showed the equilibrium dissociation constant(KD)value of 4E12-1 with PxCad TBR was 1.198×10-7 M.The cell mortality rate of 4E12-1 in the pattern of soluble protein was 24.0±10.6%(P=0.059>0.05,T-Test,n=3).The mortality rate of the larvae of P.xyllostella treated with 100 ?g/mL purified protein 4E12-1 was 10.0±1.0%(P=0.074>0.05,T-Test,n=6),there was no significant difference comparing with the blank control.In view of the low-toxicity of the mimic(VH 4E12-1),the pore formation of Bt Cry toxins was referred,and the mimic was reconstructed with Pore-forming domains in order to improve its biological activities.Through the design of different kinds of linkers,the mimic was spliced with Domain I of the Cry1Ac toxin or Domain C of colicin Ia,respectively.And the fusion proteins were expressed in E.coli and purified.Subsequently,the toxicities were identified with the sf9 cells expressing the PxCad TBR and the insect bioassays.The cytotoxicity of fusion proteins showed that the mortalities of cells for D I-4A-4E12-1 and D I-15A-4E12-1 increased by 10.3%and 20.3%comparing with the mimic 4E12-1,respectively.The results of insect bioassays showed that the mortalities of D I-4A-4E12-1 and D I-15A-4E12-1 were 18.3±7.6%(P=0.053>0.05,T-Test,n=6)and 21.7±5.8%(P=0.023<0.05,T-Test n=6),respectively,and the mortalities were significantly different compared with the mortality of 4E12-1,and the mortality of D I-15A-4E12-1 increased by 11.7%compared with that of 4E12-1.But there was no improvement in the cytotoxicity and insecticidal activity of the fusion proteins spliced with Domain C of colicin Ia.The study confirmed that the splicing of active materials of VH which target the Cry toxin receptors with the Domain I of Cry toxins can effectively improve the toxicity,while,the splicing with Ia Domain C were not.It was suspected that there were different modes of action for the different splicing products,and the appropriate pore-formation domains and splicing methods could improve the toxicity of the mimic of Cry toxin efficiently.2.Screening and activity identification for the anti-idiotype nanobody of Cry1F toxinBased on the theory of immune network,the mimic of Cry1F toxin was screened with the preparation of anti-idiotype nanobody for Cry1F toxin.The F(ab')2 fragments of Cry1F toxin polyclonal antibody were used as antigen,the anti-idiotype nanobodies(VHH)of Cry1F toxin were panned from the camelid naive antibody phage display library.After three cycles of panning,six clones were isolated and subjected for detection of the activities of binding with F(ab')2 and competitive with the Cry 1F toxin.A competition inhibition rate of 47.0±0.6%existed between the anti-idiotype nanobody(5B)and Cry1F toxin.And the 5B also bound to brush border membrane vesicles(BBMV)of O.furnacalis(Guenee)with high binding level.After the expression and purification,the competitive inhibition rate of 100 ?g/mL of the 5B soluble protein against 2 ?g/mL of Cry1F toxin could reach 45.2 ±1.6%,and the value of affinity constant(Kaff)was 1.428×10-7 L/mol.The mortality of the larvae of O.furnacalis(Guenee)treated by the 5B soluble protein compared to controls was 23.3±7.6%(P<0.05,T-Test,n=6).The anti-idiotypic nanobody has high binding activity with BBMV,but low-toxicity to target pests compared with Cry 1F toxin,while it still has the insecticidal potential to a certain extent as well as the Cry 1F toxin.3.Screening and activity analyses of the putative receptors of Cry1F toxin in Ostrinia furnacalis(Guenee)The binding proteins obtained by the Pull-Down assay were separated by the SDS-PAGE and identified through the MALDI-TOF-MS.The results showed that the biotinylated Cry1F existed around at 65 kDa,and the main bands of binding proteins appeared around at 140 to 150 kDa,95 kDa,60 kDa,50 kDa,40 kDa,37 kDa,28 kDa,17 to 20 kDa.The binding proteins include:APN,ALP,ATP-Binding Cassette(ABC)Transporters,V-ATPase,actin,and the related proteins of metabolism,signal transduction pathways and cellular components,and so on.According to the previous reports,OfAPN,OfALP and OfABCG4 were selected as the putative receptors,and the putative receptor genes were amplified and the similarities of amino acid sequences among the three putative receptors were all above 98%,compared with the sequences from the genome of O.furnacalis(Guenée).And then the recombinant proteins were expressed in E.coli BL21(DH3)and purified through Ni-chelating affinity chromatography.The binding activities of OfAPN,OfALP and OfABCG4 with Cry1F toxin were proved to be relatively effective by non-competitive ELISA and ligand blot analysis.This conclusion provided the clues for the identification of the functional receptors of the Cry1F toxin in O.furnacalis(Guenee).4.Functional identification and prediction of interaction sites between Cry1F toxin and putative receptors in Ostrinia furnacalis(Guenee)The functional receptors of the Cry1F toxin in O.furnacalis(Guenee)were identified through RNAi method.The double-strand RNA(dsRNA)of apn,alp and abcg4 from O.furnacalis(Guenée)were synthesized in vitro,and the larvae in the first-instar were fed with the dsRNA silencing the expression of the transcript of the three genes,and the relative expression levels were evaluated by the quantitative real-time PCR(qRT-PCR).Then the the RNAi treated larvae were exposed to 2.0 ?g/mL(LC80)of Cry1F toxin.After 72 h of dsRNA feeding,the inhibition rate of relative expression for gene was 45.9 ±2.7%,and that of apn and alp were 56.4± 2.3%and 60.7 ± 3.5%,respectively;the mortality rate of the treatment of apn RNAi+Cry1F was 20.0±8.7%(P=0.057>0.05,T-Test,n=6);and that of the alp RNAi+Cry1F was 11.7±5.8%(P=0.073>0.05,T-Test,n=6);the treatment of abcg4 RNAi+Cry1F resulted in a mortality rate of 30.0±8.7%(P=0.027<0.05,T-Test,n=6).The larvae of O.furnacalis(Guenée)treated with RNAi survived because of the decreases of sensitivity to Cry 1F toxin.The results showed that the OfAPN,OfALP and OfABCG4 may be the functional receptors of Cry1F toxin.The possible protein domains,transmembrane topologies and signal peptides of three putative receptors were predicted with amino acid sequences,and the constructions of phylogenetic trees were determined the genetic relationships among species.Furthermore,three-dimensional structures(3D)of Cry1F toxin and the three putative receptors were developed by homology modeling method,then the models were evaluated and optimized.The binding patterns between the Cry1F toxin and the three putative receptors via molecular docking method,and the sites of the docking complexes were predicted.The results showed that O.nubilais(Hübner)was the closest living relatives of O.furnacalis(Guenee),and the Domain ? or ? of Cry 1F toxin involved in binding with the three putative receptors,and the sites of the Cry1F-OfAPN complex might be SER311-VAL591,GLY313-TRY633,PHE315-GLN592,ARG406-TRY633,LEU451-THR527.The binding sites between Cry1F and OfALP might be ASN258-GLU306 and SER412-ARG50.And the sites of Cry1F-OfABCG4 complex might be ASN308-ARG628,PHE309-ARG628 and SER311-GLN233.It provided the references for the progress of interaction between receptors of O.furnacalis(Guenee)and Cry1F toxin,and offered the basis on screening the mimics of Cry toxins targeted and modification of structures from original Cry toxins.