Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar

Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf‐eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt‐Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non‐transgenic poplar 741 and non‐transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt‐Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt‐Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt‐Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt‐Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt‐Cry1Ac protein). The results showed that Bt‐Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt‐Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.     

A Cry1Ac Toxin Variant Generated by Directed Evolution has Enhanced Toxicity against Lepidopteran Insects

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.     

In vitro hydrolysis of Bacillus thuringiensis Cry1Ac toxin by gut proteases of Nilaparvata lugens (Stål) and binding assays of Cry1Ac toxin with brush border membrane of N. lugens midgut

Bacillus thuringiensis (Bt) and transgenic crops carrying cry genes are widely used in the management of lepidopteran and coleopteran pests. However, almost none of the Cry toxins have insecticidal properties against sap-sucking insects, such as planthoppers, leafhoppers and aphids. To understand the low insecticidal activity of Cry1Ac toxin on sap-sucking insects, we investigated two critical steps in the Bt-intoxication cascade: the proteolytic processing of Cry1Ac toxin by gut proteases, and the binding of Cry1Ac to brush border membrane vesicles (BBMV) of Nilaparvata lugens. Proteolytic processing of Cry1Ac protoxin by N. lugens gut proteases resulted in an ～65?kDa product, similar to the expected size of the trypsin-activated Cry1Ac toxin. In addition, activation of cysteine proteases in N. lugens gut increased the efficiency of proteolytic activities in the processing of Cry1Ac. However, feeding N. lugens nymphs with either Cry1Ac protoxin or trypsin-activated Cry1Ac toxin resulted in low mortalities. The LC₅₀ of Cry1Ac protoxin and trypsin-activated Cry1Ac was 198.92 and 450.18?μg/mL, respectively. In vitro binding analysis of BBMV with the pre-activated Cry1Ac showed that Cry1Ac toxin could specifically bind to the BBMV. However, binding competition with 500-fold molar excess GalNAc (N-acetyl-d-galactosamine) suggested that the binding was not mediated by GalNAc-like glycoproteins. These results indicate that Cry1Ac toxin could be successfully processed by the treatment of N. lugens gut proteases. However, the binding of Cry1Ac toxin to the midgut brush border membrane was not mediated by GalNAc-like glycoprotein. This may be responsible for the low susceptibility of N. lugens to Cry1Ac.     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.     

Partial purification of Cry 1A toxin-binding proteins from Helicoverpa armigera larval midgut

Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab. This revised version was published online in July 2006 with corrections to the Cover Date.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

Does Bt maize expressing Cry1Ac protein have adverse effects on the parasitoid Macrocentrus cingulum (Hymenoptera: Braconidae)?

The potential effects of insect‐resistant, genetically engineered (GE) crops on non‐target organisms, especially on predators and parasitoids, must be evaluated before their commercial cultivation. The effects of GE maize that produces Cry1Ac toxin on the parasitoid Macrocentrus cingulum were assessed by direct bioassay and indirect bioassay. In the indirect bioassay, parasitism rate, cocoon weight and the number of M. cingulum progeny produced per host were significantly reduced when M. cingulum‐parasitized Cry1Ac‐susceptible Ostrinia furnacalis were fed a diet containing purified Cry1Ac; however, life‐table parameters of M. cingulum were not adversely affected when the same assay was performed with Cry1Ac‐resistant O. furnacalis. These results indicated that the detrimental effects detected with a Cry1Ac‐susceptible host were mediated by poor host quality. In a direct bioassay, no difference in life‐table parameters were detected when M. cingulum adults were directly fed a 20% honey solution with or without Cry1Ac; however, survival and longevity were significantly reduced when M. cingulum adults were fed a honey solution containing potassium arsenate, which was used as a positive control. The stability and bioactivity of Cry1Ac toxin in the food sources and Cry1Ac toxin uptake by the host insect and parasitoid were confirmed by enzyme‐linked immunosorbent assay and sensitive‐insect bioassays. Our results demonstrate that M. cingulum is not sensitive to Cry1Ac toxin at concentrations exceeding those encountered in Bacillus thuringiensis maize fields. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non‐target organisms and will be useful for assessing other non‐target impacts.     

Biochemical characterization of digestive membrane‐associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis

In Brazil, the use of transgenic plants expressing the insect‐toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane‐associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in‐gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme‐linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis.     

Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.     

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins.     

The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host

Host-pathogen interactions are central components of ecological networks where the MAPK signaling pathways act as central hubs of these complex interactions. We have previously shown that an insect hormone modulated MAPK signaling cascade participates as a general switch to trans-regulate differential expression of diverse midgut genes in the diamondback moth, Plutella xylostella (L.) to cope with the insecticidal action of Cry1Ac toxin, produced by the entomopathogenic bacterium Bacillus thuringiensis (Bt). The relationship between topology and functions of this four-tiered phosphorylation signaling cascade, however, is an uncharted territory. Here, we carried out a genome-wide characterization of all the MAPK orthologs in P. xylostella to define their phylogenetic relationships and to confirm their evolutionary conserved modules. Results from quantitative phosphoproteomic analyses, combined with functional validations studies using specific inhibitors and dsRNAs lead us to establish a MAPK “road map”, where p38 and ERK MAPK signaling pathways, in large part, mount a resistance response against Bt toxins through regulating the differential expression of multiple Cry toxin receptors and their non-receptor paralogs in P. xylostella midgut. These data not only advance our understanding of host-pathogen interactions in agricultural pests, but also inform the future development of biopesticides that could suppress Cry resistance phenotypes.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.     

Antimicrobial peptides are not involved in Plutella xylostella resistance to Cry1Ac

Previous studies on Bacillus thuringiensis (Bt) resistance in the diamondback moth (DBM) Plutella xylostella have often focused on receptor proteins in the gut. In recent years, many studies have indicated that insect resistance to Bt is related to the immune system. Moreover, our group demonstrated that the expression of peptidoglycan recognition protein (PGRP) genes, which are located upstream of the insect humoral immune system signalling pathway, is significantly different between Cry1Ac-resistant and Cry1Ac-susceptible strains of DBM. Thus, whether antimicrobial peptides (AMPs), which are the major downstream effectors of the insect humoral immune system signalling pathway, are also related to the resistance of DBMs to Bt is a question worthy of in-depth study. In the present study, we sought to (a) clone the CDS of AMP genes expressed in DBMs and (b) explore the effect of the cloned AMPs on the biological characteristics and Bt resistance of DBMs. Here, we revealed three kinds of AMPs (PxDef, PxMor and PxCec), and each of their full-length CDSs was cloned. The recombinant PxDef, PxMor or PxCec proteins, which were expressed in a prokaryotic expression system, have a significant protective effect on DBMs in response to the complex microbial environment of the outside world but have no significant effect on the resistance of DBMs to Cry1Ac toxin. We believe that AMPs are not suitable as the primary entry point for studying the relationship between the immune system of DBMs and their resistance to Bt. This result will enable investigation of breakthrough ideas for further elucidating the mechanism of Bt impact on the insect immune system.     

New Insight to Structure-Function Relationship of GalNAc Mediated Primary Interaction between Insecticidal Cry1Ac Toxin and HaALP Receptor of Helicoverpa armigera

Over the last few decades Cry1Ac toxin has been widely used in controlling the insect attack due to its high specificity towards target insects. The pore-forming toxin undergoes a complex mechanism in the insect midgut involving sequential interaction with specific glycosylated receptors in which terminal GalNAc molecule plays a vital role. Recent studies on Cry toxins interactions with specific receptors revealed the importance of several amino acid residues in domain III of Cry1Ac, namely Q509, N510, R511, Y513 and W545, serve as potential binding sites that surround the putative GalNAc binding pocket and mediate the toxin-receptor interaction. In the present study, alanine substitution mutations were generated in the Cry1Ac domain III region and functional significance of those key residues was monitored by insect bioassay on Helicoverpa armigera larvae. In addition, ligand blot analysis and SPR binding assay was performed to monitor the binding characteristics of Cry1Ac wild type and mutant toxins towards HaALP receptor isolated from Helicoverpa armigera. Mutagenesis data revealed that, alanine substitutions in R511, Y513 and W545 substantially impacted the relative affinity towards HaALP receptor and toxicity toward target insect. Furthermore, in silico study of GalNAc-mediated interaction also confirmed the important roles of these residues. This structural analysis will provide a detail insight for evaluating and engineering new generation Cry toxins to address the problem of change in insect behavioral patterns.     

Polydispersity of Bacillus thuringiensis Cry1 toxins in solution and its effect on receptor binding kinetics

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.