Interaction Of Bacillus Thuringiensis Cry1A Toxin And Candidate Receptors In The Midgut Of Nilaparvata Lugens(St(?)l)

Cry toxins,produced by Bacillus thuringiensis(Bt),are used as insecticidal proteins to control many kinds of lepidopteran and coleopteran agricultural insect pests.However,the brown planthopper(BPH)Nilaparvata lugens,one of the most destructive insect pests in the rice fields of Asia,is not susceptible to general Cry toxins of Bt or transgenic rice carrying Bt cry genes.Two insecticidal mechanisms of Cry toxins were previously reported.One is the membrane insert and pore formation,while another is the signal transduction model.Specific receptors including cadherin,glycosyl-phosphatidyl-inositol(GPI)-anchored aminopeptidase N(APN),GPI-anchored alkaline phosphatase(ALP)and ABC transporter C2(ABCC2)bound to the loops of Cry toxins domain ? was one of the most important steps in the both of two mechanisms.Since that,the Cry related receptors in the midgut of BPH were analyzed.In this study,we analyzed de novo assembled transcripts from transcriptome sequencing data of BPH to identify and characterize homologs of cadherin,APN,and ALP.Then,the cadherin-,APN-and ALP-like proteins of BPH were compared to previously reported Cry binding proteins to identify their homologs in BPH.The sequence analysis revealed that at least 1 cadherin(bphCadherinl),2 APNs(bphAPN1 and bphAPN4)and 2 ALPs(bphALP4 and bphALP6)of BPH contained homologous functional domains identified from the Cry receptor associated cadherin,APN and ALP,respectively.RT-qPCR to verify the expression level of each putative Cry receptor homolog in the BPH midgut indicated that the Cry binding proteins homologous APN and ALP were expressed at high or medium high levels while the cadherin was expressed at a low level.These results suggest that homologs of Cry binding proteins exit in the midgut of BPH.Full-length sequence of the putative membrane-bound bphAPN4 was then cloned and eukaryotically expressed in a Spodoptera frugiperda(Sf9)cell line.Results of western blot,LC-MS/MS and immunofluorescence indicated that bphAPN4 was successfully prepared.However,Cytotoxicity assays showed that compared to the control cells(without overexpression of bphAPN4),overexpression of bphAPN4 in Sf9 cells did not significantly increase the cell mortality after exposure to Cry1Ac toxin.Furthermore,full-length bphAPNl,bphAPN4 and bphAPN41ackG(bphAPN4 without GPI anchor)were cloned and expressed in Drosophila melanogaster S2 cells respectively.Both bphAPNl and bphAPN4 were determined as membrane-bound proteins and were probable to anchor with the peripheral membrane through a GPI-anchored site.Only bphAPNl showed a high level of aminopeptidase activity with leucine p-nitroanilide(leu-p-NA)as the substrate.Overlay-binding analysis showed no specific binding signal when pre-activated Cry1Ac was incubated with either bphAPN1 or bphAPN4 expressed in S2 cells.No binding signal was observed by incubating bphAPN-expressed S2 cells with Cry1Ac toxin viaimmunofluorescence analysis.Cytotoxic assays showed that S2 cells expressing either bphAPN1 or bphAPN4 were not susceptible to Cry1Ac toxin.Weak binding affinity of bphAPNs with Cry toxin may account for the low susceptibility of BPH to Cry1Ac toxin.In addtion,to investigate the contribution of surface-exposed loops(loops 1,2 and 3)in the toxicity of Cry1Ab against Helicoverpa armigeralarvae,three loop-replaced Cry1Ab mutants were prokaryotically expressed in Escherichia coli BL21(DE3).Proteolytic processing of Cry1Ab toxins with trypsin indicated that the replacement of each surface-exposed loop caused no damage to toxin stability.Results of in vitro binding and competition assay indicated that,similarly to the native CrylAb toxin,the mutants retained almost the same binding affinity and specificity with brush border membrane vesicles(BBMV)of H.armigera.And toxin-overlay binding assay results showed that neither native Cry1Ab toxin nor 3 Cry mutants bound with membrane-bound bphAPNs expressed in S2 cells.Nevertheless,all Cry1Ab mutants showed less toxicity against H.armigera larvae in comparison with native CrylAb toxin.The most significant decrease in toxicity was observed from loop 2-replaced Cry1Ab mutant,with a ca 12-fold increase in the weight of tested larvae relative to that of larvae fed native CrylAb toxin.The results demonstrate that the replacement of three loops located in domain II of CrylAb toxin may result in a certain reduction in the toxicity.Relative to loop 1 and loop 3,loop 2 seems to play a more important role in generating toxicity of CrylAb against H.armigera larvae.In summary,this research was focused on the following:Cry toxins related receptor proteins were found in the midgut of BPH from transcriptome of BPH.Two membrane-bound bphAPNs(bphAPNl and bphAPN4)were expressed in Sf9 and S2 cells successfully.Relative to loop 1 and loop 3,loop 2 seems to play a more important role in generating toxicity of CrylAb against H.armigera larvae.No obvious interaction was observed on either bphAPN1 or bphAPN4 with Cry1A toxins.