Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.     

Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.     

Altered Glycosylation of 63- and 68-kilodalton microvillar proteins in Heliothis virescens correlates with reduced Cry1 toxin binding,decreased pore formation,and increased resistance to Bacillus thuringiensis Cry1 toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and 125I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.     

Binding of the CryIVD Toxin of Bacillus thuringiensis subsp. israelensis to Larval Dipteran Midgut Proteins

Ligand-blotting experiments on dipteran brush border membrane vesicles (BBMVs) showed binding of CryIVD toxin of Bacillus thuringiensis subsp. israelensis to proteins of 148 kDa in Anopheles stephensi and of 78 kDa in Tipula oleracea, both species being susceptible to CryIVD. Binding of CryIVD with BBMVs of A. stephensi resulted in a stronger signal than with BBMVs of T. oleracea. Likewise, larvae of A. stephensi are 10,000-fold more susceptible to the CryIVD toxin than are larvae of T. oleracea. Binding was also found with six proteins ranging in size from 48 to 110 kDa in BBMVs from the lepidopteran species Manduca sexta, but CryIVD was not toxic for M. sexta larvae. No binding of trypsinated CryIVD to BBMV proteins was observed. With the lepidopteran-specific toxin CryIA(b), no binding to dipteran BBMVs was found. Binding of CryIA(b) to nine different BBMV proteins ranging in size from 71 to 240 kDa was observed in M. sexta. The major binding signal was observed with a protein of 240 kDa for CryIA(b).     

Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

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.     

Altered Glycosylation of 63- and 68-Kilodalton Microvillar Proteins in Heliothis virescens Correlates with Reduced Cry1 Toxin Binding, Decreased Pore Formation, and Increased Resistance to Bacillus thuringiensis Cry1 Toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. ¹²⁵I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and ¹²⁵I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.     

Denaturation of either Manduca sexta aminopeptidase N or Bacillus thuringiensis Cry1A toxins exposes binding epitopes hidden under nondenaturing conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with (125)I-labeled Cry1Ac, Cry1Ac mutant (509)QNR-AAA(511) (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both (125)I-Cry1Ac and (125)I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots (125)I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, (125)I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. (125)I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured (125)I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) (125)I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.     

Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins

Bacillus thuringiensis Cry protein exerts its toxic effect through a receptor-mediated process. Both aminopeptidases and cadherin proteins were identified as putative Cry1A receptors from Heliothis virescens and Manduca sexta. The importance of cadherin was implied by its correlation with a Cry1Ac resistant H. virescens strain (Gahan, L. J., Gould, F., and Heckel, D. G. (2001) Science 293, 857-860). In this study, the Cry1Ac toxin-binding region in H. virescens cadherin was mapped to a 40-amino-acid fragment, from amino acids 1422 to 1440. This site overlaps with a Cry1Ab toxin-binding site, amino acids 1363-1464 recently reported in M. sexta (Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051-28056). Further, feeding of the anti-H. virescens cadherin antiserum or the partial cadherins, which contain the toxin-binding region, in combination with Cry1Ab/Cry1Ac reduced insect mortality by 25.5-55.6% to first instar H. virescens and M. sexta larvae, suggesting a critical function for this cadherin domain in insect toxicity. Mutations in this region, to which the Cry1Ac binds through its loop 3, resulted in the loss of toxin binding. For the first time, we show that the cadherin amino acids Leu(1425) and Phe(1429) are critical for Cry1Ac toxin interaction, and if substituted with charged amino acids, result in the loss of toxin binding, with a K(D) of < 10(-5) m. Mutation of Gln(1430) to an alanine, however, increased the Cry1Ac affinity 10-fold primarily due to an increase on rate. The L1425R mutant can result from a single nucleotide mutation, CTG --> CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.     

Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis

The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.     

Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.     

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.     

Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and ¹²⁵I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.     

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella)

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.