Role Of Binding Proteins In The Midgut Of Helicoverpa Armigera In Bacillus Thuringinesis Cry2Ab Insecticidal Activity

Bacillus thuringienis(Bt)is a gram-positive bacterium that synthesizes the proteinaceous crystals during sporulation that show insecticidal activity against agricultural insect pests.Due to the high specificity of each Bt strain to their target insect species and lack of toxic effect on non-target organisms,a number of Bt strains have been used as pesticides and the coding genes for their specific insecticidal proteins have been introduced to agricultural crops.After the extensive growing of Bt cotton over two decades,insect pests in the field have evolved resistance to transgenic cottons producing Cry1Ac single protein or pyramided Cry1Ac and Cry2Ab,which starts threatening the benefits and sustainable application of transgenic cotton.Understanding the mode action of the Cry proteins and the resistance mechanisms in the insects plays a crucial role in the better application of Cry proteins and to delay the evolution of insect resistance.Here we first studied the functional role of cadherin,an essential receptor of Cry1A toxins,in the mode of action of Cry2Ab.Secondly,we studied the mechanisms of resistance to Cry2Ab in Helicoverpa armigera.Functional and binding studies of Helicoverpa armigera cadherin protein in the action mechanism of Bt Cry2Ab.A region spanning cadherin repeat(CR)6 to membrane proximal region(MPR)of cadherin protein in H.armigera was targeted to detect the specific binding regions of cadherin with Cry2Ab.The target region(CR6 to MPR)was produced as six truncated cadherin peptides:CR6-8,CR7-9,CR810,CR9-11,CR10-M,and CR11-M.Ligand blot assays showed that CrylAc bound with CR9-11,CR10-M and CR11-M.In contrast,Cry2Ab bound with high affinities to CR6-8 and CR7-9 fragments and with low affinities to CR9-11,CR10-M and CR11-M fragments.ELISA assays showed that activated Cry2Ab bound specifically to both CR6-8 and CR7-9 peptides,with the dissociation constant(Kd)of 38.31±5.97 nM and 40.45±8.15 nM,respectively.Specific binding of activated Cry1Ac to CR9-11,CR10-M,and CR11-M were detected,with Kd of 2.04±0.45 nM,1.65±0.34 nM,and 0.98±0.19 nM,respectively.No specific binding of Cry2Ab to CR8-10,CR911,CR10-M,and CR11-M were observed under natural state.We transiently expressed two cadherin peptides,CR6-11,which covers the regions spanning from CR6 till the end of CR11 region,and CR6-8,with GFP as tag protein in Sf9 cells to test the functional role of cadherin.In cytotoxicity assays,Cry2Ab was not toxic to the cells expressing any of the cadherin peptides up to 1000 nM concentration.When treated with CrylAc,cells expressing only CR6-11 construct showed toxicity with EC50 value of 215.5±54.7 nM.Cry1Ac treatments,up to 1000 nM,did not affect the cells expressing CR6-8 or GFP(control)constructs.Cells transfected with full-length ABCA2 gene were highly susceptible to Cry2Ab protein,with EC50 of 113.6±29.9 nM.Cotransfection of CR6-11 peptide and ABCA2 genes in Sf9 cells,Cry2Ab showed a similar level of cytotoxicity,neither increased nor decreased activity,compared to ABCA2 alone expressing cells.However,treating ABCA2 expressing cells with different ratios of Cry2Ab and purified CR6-8 peptide together resulted significantly reduced percentages of cell death compared with those of Cry2Ab alone treatment.Silencing the cadherin and ABCA2 genes in H.armigera by RNAi,the mortality between dsGFP-and dsCAD-injected larvae showed no significant difference,the larval mortality was about 50%at 2.5 μg/g and 80%at 5.0 μg/g Cry2Ab.But the mortality reduced nearly 50%in dsABCA2-and dsCAD+dsABCA2-injected larvae,with mortality rates of 21 to 26%(2.5μg/g Cry2Ab)and 35 to 47%(5.0 μg/g Cry2Ab),respectively.Our findings prove that cadherin is not a functional receptor but could be a binding protein of Cry2Ab.Studies on the resistant mechanism of Helicoverpa armigera to Cry2Ab.A susceptible strain(96S)was selected continuously for>130 generations with sub-lethal concentrations of Cry2Ab and the resistance ratio increased to 16.2.Midgut binding studies with labeled-Cry2Ab showed significant reduction(2-fold)was observed in binding affinity of Cry2Ab to brush border membrane vesicles(BBMV)from the resistant strain(2AbR)compared with the binding to BBMV from 96S strain.Binding percentages were significantly different(P<0.05)between two strains when using lower(0.01 to 0.1 mg/ml)but not in higher concentrations(0.25 and 0.5 mg/ml)of BBMV proteins.We then used semiqualitative binding method using a fixed amount of BBMV and found no significant binding alterations in 2AbR strain compared with 96S when treated with 10 μg Cry2Ab.But bindings with 2.5 μg and 5 μg Cry2Ab,the binding percentages reduced 2-to 5-fold in 2AbR strain.Comparing binding proteins in BBMV by ligand blot,we found that five prominent proteins were consistently binding with high intensities in both strains.The protein that bound Cry2Ab with the highest intensity was at 100 kDa,that was followed by a protein approximately at 150-200 kDa.The rest three proteins,one at 45 kDa and two at 2530 kDa,bound Cry2Ab with weaker intensity than the former two proteins.Neither the high-affinity binding proteins nor their binding intensity appeared to be different between 96S BBMV and 2AbR BBMV.BBMV proteins bound to Cry2Ab were immunoprecipitated by pull-down assay and analyzed by Liquid Chromatography Mass Spectrometry(LC-MS/MS)analysis.The majority of peptides identified were matched to multiple aminopeptidase family proteins,including APN1 to APN4.ALP2 and ABCA3,homologous of ABCA2,proteins were also observed in both elute samples of 96S strain and 2AbR strain.The coding region of ABCA2 was sequenced to identify resistant alleles in 2AbR strain.We observed three different transcripts(variant a,b,and c)with missense mutation types in the resistant strain.The transcripts contained point nucleotide mutations in different exon positions.Among twelve nucleotide point-mutations,two mutations(position 137 and 191)are located in extracellular loop 1 region,one(position 566)at intracellular loop 3 between transmembrane Ⅵand ATP binding domain 1(ATP1),one(position 958)at transmembrane Ⅶ,three(position 1034,1044,and 1152)at extracellular loop 4,one(position 1263)at extracellular loop 6,and three(position 1395,1436,and 1442)between transmembrane Ⅻ and ATP-binding domain 2(ATP2)and one(position 1463)between ATP2 and transporter motifs 2(TpM2)of ATP Nucleotide Binding Fold 2(NBF2).Analyzing the transcript changes in the resistant strain by qPCR showed that expressions of APN2,APN3,APN4,APN7,ALP2,and ALP3 were significantly upregulated and of APN6 were downregulated in 2AbR strain compared to 96S strain.Upon Cry2Ab treatment to 96S strain,expessions of APN2 and APN7 were sharply reduced while those of APN1,APN3,APN4,APN6,and ALP3 were reduced.In 2AbR strain,expressions of APN3,APN4,APN7,and ALP3 were significantly reduced and those of APN1 and APN2 were increased in response to Cry2Ab treatment.APN and ALP activity assays showed that both of these enzymes were significantly reduced in BBMV of 2AbR strain,15%and 50%reduction,respectively.In contrast,5 times increased APN activities and 2 times increased ALP activities were observed in lumen of 2AbR strain.Upon continuous treatment with Cry2Ab,APN activities in BBMV of 96S strain were sharply dropped(15 to 25%)while those of 2AbR were only slightly elevated.In lumen,increased APN activities,15 to 25%,were observed in 96S strain but decreases of 10 to 70%were observed in 2AbR strain.ALP activities in BBMV of 96S strain dropped to 50-70%but those of 2AbR strain suddenly dropped to 40%at 3 h after treatment but recovered after that.ALP activities in lumen of 96S strain remained stable until about 12 h and then dropped to about 10-35%.However,in 2AbR strain,activities suddenly dropped to 60-75%with 3 h and could not recover until 48 h.Role of MAPK pathway in the mechanism of Cry2Ab resistance was further studied.Among seven MAPK member genes,the expression level of MAP3K4 was significantly upregulated>2-fold in 2AbR strain compared with 96S strain.We silenced the MAP3K4 gene by RNAi in 96S strain.At 48 h after dsMAP3K4 injection,expressions of APN2,APN3,APN7,and ALP3 were down-regulated,about 90,65,80,and 60%,respectively,and APN1 were 3-fold upregulated.Increases of APN(27%)and ALP(20%)enzyme activities were seen in BBMV of dsMAP3K4-injected larvae compared with dsGFP-injected ones.APN activity was increased 40%in lumen of dsMAP3K4-injected larvae.The primary mechanism of resistance includes reduced Cry2Ab binding to the midgut,likely associated with elevated expression levels of APN and ALP genes and their enzyme activities in the midgut BBMV and lumen.Moreover,up-regulation of a MAPK family member,MAP3K4,in the resistant strain is observed to be trans-regulating the elevation of APN and ALP genes.