APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1999, p. 1900–1903 0099-2240/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 65, No. 5
Role of Bacillus thuringiensis Toxin Domains in Toxicity and Receptor Binding in the Diamondback Moth V. BALLESTER,1 F. GRANERO,1 R. A. DE MAAGD,2 D. BOSCH,2 ´ NSUA,1 AND J. FERRE ´ 1* J. L. ME Department of Genetics, Universitat de Vale`ncia, 46100-Burjassot (Vale`ncia), Spain,1 and DLO-Centre for Plant Breeding and Reproduction Research, 6700 AA Wageningen, The Netherlands2 Received 5 November 1998/Accepted 9 February 1999
The toxic fragment of Bacillus thuringiensis crystal proteins consists of three distinct structural domains. There is evidence that domain I is involved in pore formation and that domain II is involved in receptor binding and specificity. It has been found that, in some cases, domain III is also important in determining specificity. Furthermore, involvement of domain III in binding has also been reported recently. To investigate the role of toxin domains in the diamondback moth (Plutella xylostella), we used hybrid toxins with domain III substitutions among Cry1C, Cry1E, and Cry1Ab. Neither Cry1E nor G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) was toxic, whereas Cry1C and F26 (the reciprocal hybrid) were equally toxic. H04 (a hybrid with domains I and II from Cry1Ab and domain III from Cry1C) showed toxicity that was of a similar level as that of Cry1Ab and significantly higher than that of Cry1C. Binding assays with 125I-Cry1C showed that Cry1C and F26 competed for the same binding sites on midgut membrane vesicles, whereas Cry1E, G27, and H04 did not bind to these sites. Our results show that, in contrast to findings in other insects for the toxins and hybrids used here, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I) and not by domain III. bundle of a-helices; domains II and III contain mostly b-sheets. Domain I is thought to be involved in pore formation, and domain II is thought to be involved in receptor binding and toxin specificity. The function of the C-terminal domain III was less clear for a long time, although experiments with hybrid toxins in which this domain had been exchanged showed that it can play an important role in determining the specificity of the toxin (2, 5, 13, 23). It was found that exchange of domain III can affect recognition of putative receptors on ligand blots of brush border membrane proteins (6, 20). Finally, by using different techniques, it was shown that domain III of Cry1Ac is involved in the specificity of binding to the putative Cry1Ac receptor from Manduca sexta, aminopeptidase N, as well as in the binding to intact membranes (7). Thus, it appears that at least in some insects domain III plays an important role in specificity through its involvement in specific binding to the target membranes. In the present study we used hybrid toxins with domain III substitutions in Cry1C, Cry1E, and Cry1Ab to directly test the possible role of domain III in toxin specificity and in midgut epithelial membrane binding specificity in the diamondback moth (Plutella xylostella).
From the standpoint of practical application, the characteristic of the gram-positive spore-forming bacterium Bacillus thuringiensis that makes it most interesting is the production, during sporulation, of proteinaceous crystals which are toxic to some families of insects (17, 27). B. thuringiensis toxins (also called crystal proteins) can be grouped into different classes based on sequence homology and insecticidal specificity. Of these, the best studied are the Cry1 class of crystal proteins, which are synthesized as 130-kDa protoxins and are active against Lepidoptera (17). These protoxins are solubilized in the alkaline environment of the Lepidoptera larval midgut, and then processing by midgut proteases results in a relatively stable, mature 60- to 65-kDa toxin. In susceptible insects, the activated toxins bind to the midgut epithelium and form membrane pores, which results in lysis of the epithelial cells and eventually in the death of the insect (17, 18, 27). Binding studies with purified toxins and membrane vesicles prepared from larval midguts demonstrated that the presence of receptors for a specific crystal protein is essential for toxicity and that different receptors for different crystal proteins can be present (16, 32). The importance of epithelial membrane receptors became even clearer with the observation that insects which had become resistant to one or several toxins often had lost the capacity for specific binding to these toxins because of either loss or modification of the receptors on the midgut epithelium (9, 10, 31). As can be deduced from the three-dimensional structure of the Cry3A (coleopteran-specific) and Cry1Aa (lepidopteranspecific) B. thuringiensis toxins (15, 21), toxic fragments of crystal proteins are composed of three distinct structural domains. Domain I, the most N-terminal domain, consists of a
MATERIALS AND METHODS Source of toxins. All analyses were carried out with trypsin-activated crystal proteins. Cry1C, Cry1E, Cry1Ab, and all hybrid toxins were produced in Escherichia coli XL-1 and were purified by fast protein liquid chromatography as described previously (2). Construction of the genes for hybrid toxins F26 (with domain I of Cry1C, domain II of Cry1C, and domain III of Cry1E; the domain composition is referred to herein as 1C/1C/1E), G27 (1E/1E/1C), and H04 (1Ab/1Ab/1C) is described elsewhere (2, 5). Insects and bioassays. The colony of the diamondback moth (LAB-V colony) was established from pupae collected in The Netherlands and reared in the laboratory without exposure to insecticides for more than 9 years. Insects were reared on fresh cabbage leaves at 25°C and 70 to 80% relative humidity and with a photoperiod of 16 h of light and 8 h of dark. Bioassays were performed with third-instar larvae on an artificial diet as previously described (9). Five dilutions of the toxins being tested were prepared in carbonate buffer (50 mM Na2CO3, 10 mM dithiothreitol [pH 10.0]). Fifty-microliter aliquots were applied uniformly
* Corresponding author. Mailing address: Department of Genetics, Universitat de Vale`ncia, Av. Dr. Moliner 50, 46100-Burjassot (Vale `ncia), Spain. Phone: 34-96-386-4506. Fax: 34-96-398-3029. E-mail: Juan
[email protected]. 1900
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TABLE 1. Toxicity of Cry1Ab, Cry1C, Cry1E, and some of their hybrids to third-instar larvae from a laboratory colony of P. xylostella Toxin
LC50 (FL95)a
Cry1Ab Cry1C Cry1E F26 G27 H04
15 (7–25) 117 (80–174) .1,600 83 (48–142) .1,600 6.0 (1–12)
Slope (SE)b
1.3 (0.2) 1.8 (0.2) 1.9 (0.4) 1.2 (0.3)
a
FIG. 1. Schematic representation of the structures of wild-type and hybrid toxins used in this study. Only the protease-resistant fragment is shown. Hybrids were obtained by a crossover at the junction of domains II and III.
over the surface of artificial diet dispensed in 2-cm2 wells. Carbonate buffer was used as a control for natural mortality. Each dilution was replicated two to four times with 12 to 24 larvae. Mortality was scored after 4 days, and the 50% lethal concentration (LC50; defined as the concentration required to kill 50% of the insects) and the slopes of the log dose-mortality regression line were obtained by probit analysis with the POLO computer program (26). Binding assays. Brush border membrane vesicles were prepared from lastinstar whole larvae by the differential magnesium precipitation method (8, 35) and kept at 280°C until used. Since small peptides associated to Cry1C interfere in 125I labeling (22), b-mercaptoethanol was added (final concentration, 0.1%) to a solution of Cry1C to break possible disulfide bonds between the toxin and associated peptides generated by the trypsin digestion. After incubation at room temperature for 2 h, the solution was loaded onto a Mono-Q HR5/5 column equilibrated with 20 mM Tris-HCl (pH 8.6). Cry1C was eluted with a 0 to 0.6 M NaCl gradient in 20 mM Tris-HCl (pH 8.6). Purified Cry1C (25 mg) was labeled with Na125I (0.5 mCi) by using IodoGen (Pierce) (16). Labeled Cry1C was separated from free iodine with a Bio-Gel P30 (Bio-Rad) column. Specific activity was 4.2 mCi/mg of protein, as determined by a sandwich enzyme-linked immunosorbent assay method (33). Binding experiments were performed in a final volume of 0.1 ml of binding buffer (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl [pH 7.4], 0.1% bovine serum albumin) containing 10 mg of membrane vesicle proteins, 0.2 nM 125Ilabeled Cry1C, and different concentrations of nonlabeled competitor. Incubations were carried out at room temperature for 90 min. Bound and free 125Ilabeled Cry1C proteins were separated by filtration through GF/F glass fiber filters (Whatman) presoaked in binding buffer with 0.5% bovine serum albumin. Filters were washed with 5 ml of cold binding buffer, and the radioactivity retained was measured in a 1282 Compugamma CS gamma counter (LKB). Nonspecific binding was determined by adding a 500-fold excess of nonlabeled Cry1C. Maximum specific binding was about 3% of total radioactivity. Replicate data from a single batch of brush border membrane vesicles were analyzed with the LIGAND computer program (25).
RESULTS Toxicity of wild-type and hybrid toxins. Involvement of toxin domains in toxin specificity was studied with the wild-type toxins Cry1Ab, Cry1C, and Cry1E, as well as with hybrid toxins, between Cry1C and Cry1Ab (H04 [1Ab/1Ab/1C]) and between Cry1C and Cry1E (F26 [1C/1C/1E] and G27 [1E/1E/1C]) (Fig. 1). The wild-type toxins differed considerably in toxicity against the diamondback moth, with Cry1Ab being the most active toxin (LC50 5 15 ng/cm2 of artificial diet), Cry1C being moderately active (LC50 5 117 ng/cm2), and Cry1E being very slightly toxic or not toxic at all (LC50 . 1,600 ng/cm2) (Table 1). Among the hybrid toxins, those with domains I and II from Cry1Ab (H04) or from Cry1C (F26) showed toxicity against the diamondback moth, whereas the hybrid toxin with domains I and II from Cry1E (G27) did not (Table 1). Moreover, LC50s of the hybrid toxins were not significantly different from those of the respective wild-type toxins from which their domains I and II were derived. Toxins carrying the same domain III (such as either Cry1E and F26 or G27, Cry1C, and H04) did not show any similarity in terms of toxicity. Binding of Cry1C to brush border membrane vesicles and competition studies. Binding of 125I-labeled Cry1C was com-
LC50s and 95% fiducial limits (FL95) are expressed as nanograms of protein per square centimeter of artificial diet. b Slope of the probit regression line.
peted effectively by nonlabeled Cry1C and by F26 but not by G27 or H04 (Fig. 2). Thus, only those toxins carrying the same domains I and II and not those carrying the same domain III competed for the same binding sites. The dissociation constant and concentration of binding sites for Cry1C (Kd 5 7.1 6 1.1 nM; Rt 5 7.2 6 1.6 pmol/mg of vesicle protein) and for F26 (Kd 5 7.2 6 0.7 nM; Rt 5 8.7 6 1.6 pmol/mg of vesicle protein) were essentially identical. Replacement of domain III of Cry1C by that of Cry1E (in hybrid F26) did not affect its binding parameters, either qualitatively or quantitatively. DISCUSSION The information provided by studies on high-resolution three-dimensional structures of Cry3A and Cry1Aa toxins (15, 21), site-directed mutagenesis (12, 19), and the correlation between cross-resistance and sequence similarity of B. thuringiensis toxins (29) strongly suggests that domain II is involved in binding and/or receptor interaction in the midgut epithelial membrane and thus is one major determinant of toxin specificity. Similarly, there is strong experimental evidence that domain I is involved in membrane insertion to form the pore (4, 11, 34). However, the role of domain III has only recently become clearer. Domain-swapping experiments showed that substitution of domain III of a slightly active toxin, such as that of Cry1Aa for Heliothis virescens by that of the very active Cry1Ac, can increase considerably its toxicity to that insect (13). Cry1Ab and Cry1E, which are both inactive for Spodoptera exigua, can be rendered toxic for this insect by replacing their domains III by that of the active Cry1C (2, 5). For S. exigua, this resulted in hybrid toxins that were even more active than their parental toxin Cry1C (6). Thus, at least in these insects, domain III is also an important determinant of toxin specificity. Several functions for domain III at the molecular level have been proposed. It was suggested that domain III confers stability on the toxin molecule (21). Mutagenesis experiments have shown that it may be involved in pore formation (3, 36). More recently it was shown that domain III exchange may change the recognition of putative receptors on ligand blots of separated brush border membrane vesicle proteins of several insects (5, 6, 20), suggesting that domain III has a function in binding. Finally, domain III of Cry1Ac was shown to be involved in specific binding to the putative receptor aminopeptidase N of M. sexta, as well as to intact brush border membrane vesicles, and in both cases domain III was involved in the inhibition of binding by the sugar N-acetylgalactosamine (7). Thus, it appears that in at least some insects domain III plays a role in determining specificity through its involvement in high-affinity binding. To determine which of the above-mentioned roles of do-
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FIG. 2. Binding of 125I-labeled Cry1C to brush border membrane vesicles at increasing concentrations of nonlabeled competitor. h (broken line), Cry1C; E (solid line), F26; ■, H04; F, G27.
main III are applicable to the diamondback moth, we tested a series of hybrid toxins with domains I and II from one toxin and domain III from another. As in S. exigua and Mamestra brassicae, Cry1E has little or no toxicity to the diamondback moth. In contrast to the results for the former two insects, we found that domain III substitution in Cry1E has no effect on toxicity against the diamondback moth, since G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) is not toxic. Whereas Cry1E binding was demonstrated for S. exigua, the diamondback moth seems to lack high-affinity midgut receptors for Cry1E (24). This suggests that the lack of toxicity of Cry1E, as well as of G27, is caused by the absence of an appropriate domain II functioning in binding. The complementary hybrid F26, which is Cry1C with its domain III replaced with that of Cry1E, is as toxic as Cry1C. Likewise, replacement of domain III of Cry1Ab by that of Cry1C (H04) does not alter toxicity. These results suggest that given functional domains I and II, domains III of Cry1C, Cry1E, and Cry1Ab are interchangeable. Heterologous competition analyses consist of measuring the binding of a labeled molecule at increasing concentrations of other nonlabeled molecules and may be used to determine if two different molecules bind to the same receptor. In the diamondback moth, Cry1Ab and Cry1C have been shown to bind to different receptors, since increasing concentrations of the former do not affect binding of labeled Cry1C (9) and vice versa (14). Also, because Cry1E lacks receptors in this insect (24), no competition with Cry1C is expected. Heterologous competition analyses with labeled Cry1C and hybrid toxins with domain III replacements between Cry1C and either Cry1E or Cry1Ab showed that binding to brush border membrane vesicles from the diamondback moth was not affected by domain III replacement. Hybrid toxins with domain III from Cry1C (G27 and H04) did not compete for binding with Cry1C, whereas the hybrid toxin with domain II (and I) from Cry1C (F26) did compete (Fig. 2). The dissociation constant and binding site concentration for Cry1C and F26 were essentially identical, and their values did not differ significantly from those obtained for Cry1C in previous studies: Kd 5 6.5 6 0.8 nM and Rt 5 10.8 6 3.3 pmol/mg of vesicle protein (9), Kd 5 8.8 6 0.3 nM and Rt 5 3.2 6 0.0 pmol/mg of vesicle protein (28), and Kd
5 8.9 6 0.1 nM and Rt 5 9.2 6 1.0 pmol/mg of vesicle protein (37). Moreover, in a study on a Dipel-resistant diamondback moth colony from Hawaii, it was shown that this colony was resistant to Cry1Ab, Cry1Ac, and H04 but not to Cry1C (29). The resistance to Cry1A toxins was shown to be correlated with reduced binding of Cry1Ab and Cry1Ac (1, 30). These and the results presented here indicate that the specificity of binding in the diamondback moth is mediated by domain II (and/or I) but not by domain III. Therefore, in contrast to findings in other insects for the toxins and hybrids used in this study, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I), with the domain III pairs 1C-1E and 1C-1Ab being mutually interchangeable. If domain III plays a role at all in binding or toxicity in the diamondback moth, it is less specific than in other cases. ACKNOWLEDGMENTS We thank Petra Bakker for preparing and purifying the crystal proteins. This work was supported by a grant from the European Union under the ECLAIR program (project no. AGRE-0003) and a grant from the Spanish Ministerio de Agricultura, Pesca y Alimentacio ´n (project no. AGR91-0238-CE). REFERENCES 1. Ballester, V., F. Granero, B. E. Tabashnik, T. Malvar, and J. Ferre´. 1999. Integrative model for binding of Bacillus thuringiensis toxins in susceptible and resistant larvae of the diamondback moth (Plutella xylostella). Appl. Environ. Microbiol. 65:1413–1419. 2. Bosch, D., B. Schipper, H. Van der Kleij, R. A. De Maagd, and W. J. Stiekema. 1994. Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/Technology 12:915– 918. 3. Chen, X. J., M. K. Lee, and D. H. Dean. 1993. Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts. Proc. Natl. Acad. Sci. USA 90:9041–9045. 4. Cummings, C. E., G. Armstrong, T. C. Hodgman, and D. J. Ellar. 1994. Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of a Bacillus thuringiensis d-endotoxin. Mol. Membr. Biol. 11:87–92. 5. de Maagd, R. A., M. S. G. Kwa, H. van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema, and D. Bosch. 1996. Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for
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VOL. 65, 1999
6.
7.
8.
9.
10. 11. 12. 13.
14. 15. 16.
17. 18. 19. 20. 21. 22.
Spodoptera exigua and altered membrane protein recognition. Appl. Environ. Microbiol. 62:1537–1543. de Maagd, R. A., H. van der Klei, P. L. Bakker, W. J. Stiekema, and D. Bosch. 1996. Different domains of Bacillus thuringiensis d-endotoxins can bind to insect midgut membrane proteins on ligand blots. Appl. Environ. Microbiol. 62:2753–2757. De Maagd, R. A., P. L. Bakker, L. Masson, M. J. Adang, S. Sangadala, W. Stiekema, and D. Bosch. 1999. 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. Mol. Microbiol. 34:463– 471. Escriche, B., F. J. Silva, and J. Ferre´. 1995. Testing suitability of brush border membrane vesicles prepared from whole larvae from small insects for binding studies with Bacillus thuringiensis CryIA(b) crystal protein. J. Invertebr. Pathol. 65:318–320. Ferre´, J., M. D. Real, J. Van Rie, S. Jansens, and M. Peferoen. 1991. Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor. Proc. Natl. Acad. Sci. USA 88:5119–5123. Ferre´, J., B. Escriche, Y. Bel, and J. Van Rie. 1994. Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins. FEMS Microbiol. Lett. 132:1–7. Gazit, E., and Y. Shai. 1995. The assembly and organization of the a5 and a7 helices from the pore-forming domain of Bacillus thuringiensis d-endotoxin: relevance to a functional model. J. Biol. Chem. 270:2571–2578. Ge, A. Z., N. I. Shivarova, and D. H. Dean. 1989. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis d-endotoxin protein. Proc. Natl. Acad. Sci. USA 86:4037–4041. Ge, A. Z., D. Rivers, R. Milne, and D. H. Dean. 1991. Functional domains of Bacillus thuringiensis insecticidal crystal proteins: refinement of Heliothis virescens and Trichoplusia ni specificity domains on CryIA(c). J. Biol. Chem. 266:17954–17958. Granero, F., V. Ballester, and J. Ferre´. 1996. Bacillus thuringiensis crystal proteins Cry1Ab and Cry1Fa share a high affinity binding site in Plutella xylostella. Biochem. Biophys. Res. Commun. 224:779–783. Grochulski, P., L. Masson, S. Borisova, M. Pusztai-Carey, J. L. Schwartz, R. Brousseau, and M. Cygler. 1995. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation. J. Mol. Biol. 254:447–464. Hofmann, C., H. Vanderbruggen, H. Ho ¨fte, J. Van Rie, S. Jansens, and H. Van Mellaert. 1988. Specificity of Bacillus thuringiensis d-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc. Natl. Acad. Sci. USA 85:7844– 7848. Ho ¨fte, H., and H. R. Whiteley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53:242–255. Knowles, B. H., and J. A. T. Dow. 1993. The crystal d-endotoxins of Bacillus thuringiensis: models for their mechanism of action on the insect gut. Bioessays 15:469–476. Lee, M. K., R. E. Milne, A. Z. Ge, and D. H. Dean. 1992. Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis d-endotoxin. J. Biol. Chem. 267:3115–3121. Lee, M. K., B. A. Young, and D. H. Dean. 1995. Domain III exchanges of Bacillus thuringiensis CryIA toxins affect binding to different gypsy moth midgut receptors. Biochem. Biophys. Res. Commun. 216:306–312. Li, J., J. Carroll, and D. J. Ellar. 1991. Crystal structure of insecticidal d-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. Nature 353:815– 821. Luo, K., and M. J. Adang. 1994. Removal of adsorbed toxin fragments that
23.
24.
25. 26. 27. 28. 29.
30.
31. 32.
33.
34.
35.
36.
37.
1903
modify Bacillus thuringiensis CryIC d-endotoxin iodination and binding by sodium dodecyl sulfate treatment and renaturation. Appl. Environ. Microbiol. 60:2905–2910. Masson, L., A. Mazza, L. Gringorten, D. Baines, V. Aneliunas, and R. Brousseau. 1994. Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system. Mol. Microbiol. 14:851–860. Masson, L., A. Mazza, R. Brousseau, and B. E. Tabashnik. 1995. Kinetics of Bacillus thuringiensis toxin binding with brush border membrane vesicles from susceptible and resistant larvae of Plutella xylostella. J. Biol. Chem. 270:11887–11896. Munson, P. J., and D. Rodbard. 1980. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220–239. Rusell, R. M., J. L. Robertson, and N. E. Savin. 1977. POLO: a new computer program for probit analysis. Bull. Entomol. Soc. Am. 23:209–213. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775–806. Tabashnik, B. E., N. Finson, F. R. Groeters, W. J. Moar, M. W. Johnson, K. Luo, and M. J. Adang. 1994. Reversal of resistance to Bacillus thuringiensis in Plutella xylostella. Proc. Natl. Acad. Sci. USA 91:4120–4124. Tabashnik, B. E., T. Malvar, Y.-B. Liu, N. Finson, D. Borthakur, B.-S. Shin, S.-H. Park, L. Masson, R. A. de Maagd, and D. Bosch. 1996. Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 62:2839–2844. Tabashnik, B. E., Y.-B. Liu, T. Malvar, D. G. Heckel, L. Masson, V. Ballester, F. Granero, J. L. Me´nsua, and J. Ferre´. 1997. Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis. Proc. Natl. Acad. Sci. USA 94:12780–12785. Van Rie, J., W. H. McGaughey, D. E. Johnson, B. D. Barnett, and H. Van Mellaert. 1990. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science 247:72–74. Van Rie, J., S. Jansens, H. Ho¨fte, D. Degheele, and H. Van Mellaert. 1990. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl. Environ. Microbiol. 56:1378–1385. Voller, A., D. Bidwell, and A. Barlett. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections, p. 506–512. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. Von Tersch, M. A., S. L. Slatin, C. A. Kulesza, and L. H. English. 1994. Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide. Appl. Environ. Microbiol. 60:3711–3717. Wolfersberger, M., P. Luethy, A. Maurer, P. Parenti, F. V. Sacchi, B. Giordana, and G. M. Hanozet. 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A:301–308. Wolfersberger, M. G., X. J. Chen, and D. H. Dean. 1996. Site-directed mutations in the third domain of Bacillus thuringiensis d-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles. Appl. Environ. Microbiol. 62:279–282. Wright, D. J., M. Iqbal, F. Granero, and J. Ferre´. 1997. A change in a single midgut receptor in the diamondback moth (Plutella xylostella) is only in part responsible for field resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. Appl. Environ. Microbiol. 63:1814–1819.
