Bacillus Thuringiensis Cry1Ac Toxin Research Articles

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Significant sensing performance of an all-silicon terahertz metasurface chip for Bacillus thuringiensis Cry1Ac protein

The promising prospect of a terahertz metasurface in sensing and detection applications has attracted increasing attention because of its ability to overcome the classical diffraction limit and the enhancement of field intensity. In this work, a novel scheme based on an all-silicon terahertz plasmon metasurface is proposed and experimentally demonstrated to be a highly sensitive biosensor for the Bacillus thuringiensis Cry1Ac toxin. The regression coefficients between Bacillus thuringiensis protein concentrations and the spectral resonance intensity and frequency were 0.8988 and 0.9238, respectively. The resonance amplitude variation and frequency shift of the metasurface were investigated in terms of both thickness and permittivity change of the analyte, which reflected the protein residue in the actual process. Moreover, the reliability and stability of the metasurface chip were verified by time period, temperature, and humidity control. These results promise the ability of the proposed metasurface chip as a Bacillus thuringiensis protein sensor with high sensitivity and stability. In addition, this novel device strategy provides opportunities for the advancement of terahertz functional applications in the fields of biochemical sensing and detection.

Frequency of resistance alleles to Cry1Ac toxin from cotton bollworm, Helicoverpa armigera (Hübner) collected from Bt-cotton growing areas of Telangana state of India

Transgenic cotton expressing Bacillus thuringiensis (Bt) cry1Ac and cry2Ab toxin genes is widely cultivated to manage bollworm complex in India. Cotton bollworm Helicoverpa armigera (Hübner) is one of the most serious of this complex. It is likely to evolve resistance to Cry toxins in view of continual selection pressure due to extensive cultivation of Bt cotton. Monitoring susceptibility of cotton bollworm using conventional bioassays is reported to have shown its increasing tolerance to Cry1Ac over the years. We report using an F2 screen Cry1Ac resistance allele frequencies of 0.050 (95% CI 0.022–0.076) and 0.056 (95% CI 0.035–0.075) in the insect populations collected from pigeon pea grown alongside Bt cotton in the respective years of 2016 and 2017 in the Telangana state of India. Compared to our earlier studies for 2013 and 2014, resistance allele frequency to Cry1Ac in the cotton bollworm in the following two years remains unchanged. The significance of these results is discussed in the context of non-Bt host crops acting as refuge for cotton bollworm for ensuring sustainable resistance management.

GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells.

The Cadherin Cry1Ac Binding-Region is Necessary for the Cooperative Effect with ABCC2 Transporter Enhancing Insecticidal Activity of Bacillus thuringiensis Cry1Ac Toxin.

Bacillus thuringiensis Cry1Ac toxin binds to midgut proteins, as cadherin (CAD) and ABCC2 transporter, to form pores leading to larval death. In cell lines, co-expression of CAD and ABCC2 enhance Cry1Ac toxicity significantly, but the mechanism remains elusive. Here, we show that the expression of Helicoverpa armigera CAD (HaCAD-GFP) in Hi5 cells induces susceptibility to Cry1Ac and enhanced Cry1Ac toxicity when co-expressed with H. armigera ABCC2 (HaABCC2-GFP), since Cry1Ac toxicity increased 735-fold compared to Hi5 cells expressing HaCAD-GFP alone or 28-fold compared to HaABCC2-GFP alone. In contrast, the expression of the Spodoptera litura CAD (SlCAD-GFP) in Hi5 cells did not induce susceptibility to Cry1Ac nor it potentiated Cry1Ac toxicity with HaABCC2-GFP. To identify the CAD regions involved in the enhancement of Cry1Ac toxicity with ABCC2, the different CAD domains were replaced between SlCAD-GFP and HaCad-GFP proteins, and cytotoxicity assays were performed in Hi5 cells in the absence or presence of HaABCC2-GFP. The HaCAD toxin-binding region (TB), specifically the CAD repeat-11, was necessary to enhance Cry1Ac toxicity with ABCC2. We propose that CAD TB is involved in recruiting Cry1Ac to localize it in a good position for its interaction with the ABCC2, resulting in efficient toxin membrane insertion enhancing Cry1Ac toxicity.

ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

Fitness Cost Associated with Resistance to Bacillus thuringiensis Cry1ac Toxin in Helicoverpa armigera (Hübner)

Transgenic cotton producing a Bacillus thuringiensis (Bt) Cry1Ac toxin is widely used for controlling the cotton bollworm, Helicoverpa armigera . The lessons learnt from the usage of insecticides suggest deployment of effective resistance management strategies to preserve the long-term utility of Bt-cotton. Consequently, it is important to understand the interaction of Cry1Ac toxin with distinct populations of the resistant alleles (homozygote resistant RR, susceptible SS and heterozygote RS or SR) keeping in mind the fitness cost associated with resistance. The present studies were under taken to understand the in vitro response of all such allelic populations. A critical analysis on the effects of Bt-toxin on different development stages shows that irrespective of the allelic genotype, the toxin exerts inhibitory influence on all the developmental stages. This effect is visualized as an enormous decrease in larval, pupal and adult weight, wing expanse of adults, adult life span and sex-ratio that was coupled with increase in time taken to pupate, pupal duration and total developmental period. Majority of the emerged adults possessed different types of abnormalities (wingless, deformed wings). They did not mate to lay eggs and if eggs were laid, they normally did not hatch, thus resulting in total loss of population(s). Cry1Ac toxin exerts a high cost of fitness on H. armige ra and in this context susceptible and heterozygous genotypes were the most affected.

Amino acids Y229 and F603 are involved in Bacillus thuringiensis Cry1Ac δ-endotoxin stability and toxicity

Bacillus thuringiensis Cry1Ac toxin shares structurally five conserved blocs with the other δ-endotoxins. To study the role of some amino acids belonging to these regions, two mutations, Y(229) P and F(603) S, were introduced respectively in blocs 2 and 5. The stability and crystallization of the resulting mutant proteins Cry1Ac'1 and Cry1Ac'3 were affected. Both of them lost their toxicity to the Lepidopteran larvae Ephestia kuehniella. Unlike Cry1Ac'1, Cry1Ac'3 became very sensitive to proteases. Accordingly, the three-dimensional structures of the two mutants were studied. The obtained models showed that both of the residues, Y229, located near the bottom of the α7 helix, and F603, located in the core of domain III, are involved in hydrophobic interactions essential for protein stability and toxicity. These results reveal that conserved amino acids blocs of Cry toxins have conformational and functional roles.

Susceptibility of Helicoverpa armigera from different host plants in northern China to Bacillus thuringiensis toxin Cry1Ac

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a serious pest of cotton and many other crops in northern China. To evaluate the contribution of alternative hosts as an effective refuge for transgenic cotton expressing the Bacillus thuringiensis (Bt) Cry1Ac toxin, the susceptibility to this toxin was measured in progeny derived from field-collected H. armigera larvae and pupae from different hosts in the Xiajin’s region of the Shandong Province in northern China. During 2008–2010, progeny from a total of 258,56,184 and 160 single-pair crosses derived from wheat (first-generation), Bt cotton (second-generation), Bt cotton (third-generation), and corn (third-generation) were screened on Cry1Ac diets, respectively. Based on relative average development rates (RADR) of H. armigera larvae in these F 1 tests, the second and third-generation moths emerging from Bt cotton fields were more tolerant to the Bt toxin than the first and third-generation moths emerging from wheat and corn each year. These results suggest that there is significant variation in susceptibility to Bt toxins among H. armigera populations derived from different host crops. Alternate crops, such as corn, that maintain Bt susceptible populations of H. armigera could be used as refugia to minimize the evolution of resistance to Bt cotton.

Entomopathogenicity of endophytic Serratia marcescens strain SRM against larvae of Helicoverpa armigera (Noctuidae: Lepidoptera)

An endophytic Serratia marcescens strain SRM (MTCC 8708) isolated from the flowers of summer squash was found to be entomopathogenic against the larvae of Helicoverpa armigera. Natural epizootic of this bacterial strain on the larvae collected from summer squash flowers ranged from 19.9 to 72.3%. Under laboratory conditions, a dose of 6 × 1010 c.f.u./ml diet induced 66.3% mortality of first instar H. armigera larvae. Similarly all other growth and development parameters of the insect were severely retarded in a dose-dependent manner. The bacterium invaded the entire alimentary canal and haemolymph with successful replacement of all other gut-associated microflora. There was a great reduction in midgut proteinase activity due to inhibition of five major proteinase isozymes by S. marcescens infection. Further, a synergistic interaction between chitinases isolated from this strain and Bacillus thuringiensis Cry1Ac toxin was observed. The present findings suggest that this plant-associated S. marcescens strain SRM could be suitably exploited for the management of H. armigera.

Vip3Aa Tolerance Response of Helicoverpa armigera Populations From a Cry1Ac Cotton Planting Region

Transgenic cotton, Gossypium hirsutum L., that expresses the Bacillus thuringiensis (Bt) Cry1Ac toxin, holds great promise in controlling target insect pests. Evolution of resistance by target pests is the primary threat to the continued efficacy of Bt cotton. To thwart pest resistance evolution, a transgenic cotton culitvar that produces two different Bt toxins, cry1Ac and vip3A genes, was proposed as a successor of cry1Ac cotton. This article reports on levels of Vip3Aa tolerance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) populations from the Cry1Ac cotton planting region in China based on bioassays of the F1 generation of isofemale lines. In total, 80 isofemale families of H. armigera from Xiajin county of Shandong Province (an intensive Bt cotton planting area) and 93 families from Anci county of Hebei Province (a multiple-crop system including corn [Zea mays L.] , soybean [Glycine max (L.) Merr.], peanut (Arachis hypogaea L.), and Bt cotton) were screened with a discriminating concentration of both Cry1Ac- and Vip3A-containing diets in 2009. From data on the relative average development rates and percentage of larval weight inhibition of F1 full-sib families tested simultaneously on Cry1Ac and Vip3Aa, results indicate that responses to Cry1Ac and Vip3Aa were not genetically correlated in field population ofH. armigera. This indicates that the threat of cross-resistance between Cry1Ac and Vip3A is low in field populations of H. armigera. Thus, the introduction of Vip3Aa/Cry1Ac-producing lines could delay resistance evolution in H. armigera in Bt cotton planting area of China.

Residue 544 in Domain III of the Bacillus thuringiensis Cry1Ac Toxin is Involved in Protein Structure Stability

A unique residue W544 in the beta18-beta19 loop of the Bacillus thuringiensis Cry1Ac toxin has been implicated in its toxicity. In this study, the effects of mutations at this residue on protein stability during protease treatment, UV irradiation, and preservation were examined. Residue 544 of Cry1Ac was involved in maintaining structural stability, and substitution of a polar group at this position was unfavorable to protein stability. One mutant, W544F, produced larger crystals and was more stable. This mutant showed greater resistance to UV radiation than the wild type Cry1Ac but retained equal toxicity. This is the first report showing that residue 544 in the Cry1Ac domain III plays a significant role in toxin structural stability. Our W544F mutant is a significant development in terms of field applications of Cry1Ac toxin.

Regulation of the seasonal population patterns of Helicoverpa armigera moths by Bt cotton planting

Transgenic cotton expressing the Bacillus thuringiensis (Bt) Cry1Ac toxin has been commercially cultivated in China since 1997, and by 2000 Bt cotton had almost completely replaced non-transgenic cotton cultivars. To evaluate the impact of Bt cotton planting on the seasonal population patterns of cotton bollworm, Helicoverpa armigera, the dynamics of H. armigera moths were monitored with light traps from four locations (Xiajin, Linqing and Dingtao of Shandong Province; Guantao of Hebei Province) in high Bt density region and five locations (Anci and Xinji of Hebei Province; Dancheng and Fengqiu of Henan Province; Gaomi of Shandong Province) in low Bt density region from 1996 to 2008. A negative correlation was found between moth densities of H. armigera and the planting years of Bt cotton in both high and low Bt density areas. These data indicate that the moth population density of H. armigera was reduced with the introduction of Bt cotton in northern China. Three generations of moths occurred between early June and late September in the cotton regions. Interestingly, second-generation moths decreased and seemed to vanish in recent years in high Bt density region, but this tendency was not found in low Bt density region. The data suggest that the planting of Bt cotton in high Bt density region was effective in controlling the population density of second-generation moths. Furthermore, the seasonal change of moth patterns associated with Bt cotton planting may regulate the regional occurrence and population development of this migratory insect.

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.

N546 in β18–β19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin

Our previous mutagenic analysis showed that the unique residue N546 in the apex of β18–β19 loop of Bacillus thuringiensis Cry1Ac toxin is important for its toxicity. In this study, trypsin digestion susceptibility, binding to BBMV and oligomer formation activity was therefore analyzed to determine the mechanism of toxicity change of these mutant toxins. The results showed that residue N546 was not involved in toxin oligomerisation and maintaining the stability of toxin, the enhanced toxicity of mutant N546A was just because of increased binding to BBMV, and reduction in toxicity of other mutants were caused by reduction in initial or irreversible binding to BBMV. This is the first report that revealed N546 in Cry1Ac domain III played an essential role in its insecticidal activity and binding to insect BBMV.

Tritrophic impacts of Bt-expressing transgenic pine on the parasitoid Meteorus pulchricornis (Hymenoptera: Braconidae) via its host Pseudocoremia suavis (Lepidoptera: Geometridae)

Pseudocoremia suavis is a New Zealand endemic geometrid present in commercial pine plantations where it has occasionally reached pest levels. It is parasitized by a self-introduced, generalist, solitary, braconid endoparasitoid, Meteorus pulchricornis. Tritrophic impacts on M. pulchricornis of transgenic pine, Pinus radiata, expressing Bacillus thuringiensis (Bt) Cry1Ac toxin, were examined using a laboratory-based system with P. suavis as the host. Unparasitized P. suavis fed with Bt-expressing pine foliage had significantly higher mortality than those fed with control pine and surviving larvae had reduced growth rates and delayed pupation. Parasitoid survival from Bt pine-fed hosts was reduced significantly when pre- and post-emergence survival was combined. Observed pre-emergence host mortality in parasitized Bt pine-fed P. suavis was consistent with the level of host mortality expected if parasitism, which itself caused significant pre-emergence host death, and the Bt toxin were acting independently as larval mortality factors. Parasitized hosts fed on Bt pine had significantly reduced growth rates and parasitoids took longer to emerge. The degree of developmental delay caused by Bt appeared to be greater for unparasitized hosts than for parasitoids. There was no effect of Bt on the parasitoid pupal duration, weight of pupa in cocoon, or number of eggs in F 1 adults’ ovaries. Across both treatments, the number of days to parasitoid emergence was negatively correlated with host weight on parasitism day, and the number of eggs in F 1 adults’ ovaries was positively correlated with cocoon weight.

Effects of Bt transgenic Chinese cabbage pollen expressing Bacillus thuringiensis Cry1Ac toxin on the non-target insect Bombyx mori (Lepidoptera: Bombyxidae) larvae

We evaluated the effects of the pollens of transgenic Chinese cabbage ( Brassica campestris subsp. napus var. pekinensis Makino), expressing Bacillus thuringiensis Cry1Ac toxin ( Bt cabbage), on Bombyx mori larvae. Decreased survival rate and body weight of B. mori larvae were observed when fed with an artificial diet containing Bt cabbage pollens. ELISA test using Cry1Ac-antibody revealed that the Cry toxin was detected not only in the alimentary canal but also in the hemolymph and remaining body parts, suggesting that the ingested Cry1Ac protein is distributed inside the body likely through the damaged midgut epithelial membrane of B. mori. Taken together, our results suggested that Bt cabbage pollens adversely affect non-target insect B. mori larvae when consumed. Considering the low possibility of Bt cabbage pollen exposure to B. mori in the actual field condition, however, the risk would be minimal.

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.

The mutation R 423S in the Bacillus thuringiensis hybrid toxin CryAAC slightly increases toxicity for Mamestra brassicae L.

Bacillus thuringiensis Cry1Ac toxin is 100 times less toxic than Cry1C to Mamestra brassicae. An R 423S mutation abolishes Cry1Ac toxin proteolysis in M. brassicae gut juice but does not increase its toxicity to this insect. The CryAAC hybrid toxin (1Ac/1Ac/1Ca) is toxic to M. brassicae but is susceptible to gut protease digestion at the R 423 residue. Accordingly we have investigated the effect of the R 423S mutation in CryAAC on its toxicity for M. brassicae and Pieris brassicae. Bioassays demonstrated that the R 423S mutation slightly increased the toxicity of CryAAC for M. brassicae by having a significantly inhibitory effect on the growth of surviving larvae. The mutant hybrid was still highly toxic to P. brassicae. Features of CryAACR 423S such as, (1) stability in M. brassicae gut juice and (2) crystal solubility were investigated. Computer simulations suggest that a possible major increase in flexibility in the CryAAC loop β7/β8 (G 391–P 397) caused by the R 423S substitution could be a reason for the increase in M. brassicae toxicity.

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to the 120 kDa APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analysed the various glycans on the 120 kDa APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of the 4 possible N-glycosylation sites are occupied with fucosylated paucimannose {Man 2–3(Fuc 1–2)GlcNAc 2-peptide} type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal “stalk” region of the protein. It is likely that 5–6 of the 13 sites are occupied, probably with simple {GalNAc-peptide} type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc rich, is the most likely binding site for Cry1Ac.