Toxicity and Mode of Action of Bacillus thuringiensis Cry Proteins in the Mediterranean Corn Borer, Sesamia nonagrioides (Lefebvre)

Abstract

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with ¹²⁵I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC₃(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.

The Mediterranean corn borer, Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), is one of the most damaging pests of corn in Spain and other Mediterranean countries (12). Chemical control is particularly difficult because the larvae tunnel throughout the stem from the first instar. Genetically engineered corn plants expressing δ-endotoxins from Bacillus thuringiensis (Bt corn) can effectively control this major corn pest and at the same time can reduce the environmental costs associated with the use of conventional insecticides (29).

Bt corn is commercially grown in nine countries and occupied a global surface area of 11.2 × 10⁶ ha in 2004 (18). The cultivated area containing Bt corn has been steadily increasing in Spain since its commercialization in 1998 and reached about 58,000 ha in 2004. Development of resistance within target insect populations is becoming the main threat for the long-term success of this technology (3). Bt corn expressing the Cry1Ab toxin is being widely used for the control of corn borers, and commercialization of corn expressing the Cry1Fa protein has recently been approved (Pioneer/Mycogen Seeds).

Several strategies to delay the development of resistance have been devised, and some of these strategies are currently mandatory (http://www.epa.gov/pesticides/biopesticides). Mathematical modeling and greenhouse assays have shown that expression of different toxins in the same plant (pyramiding) substantially increases the time required for development of resistance in the exposed populations (27, 37). For this strategy to be effective it is necessary that there is an absence of cross-resistance to the pyramided toxins. A number of studies have shown that if several toxins share binding sites in the larval midgut, it is likely that once the insects develop resistance to one of them, they become resistant to the others (2, 11, 20).

The aims of the present study were to determine the mode of action of the Cry1Ab protein on S. nonagrioides and to search for other Cry proteins that could be used to control this pest and that could be compatible in pyramided transgenic plants for better management of insect resistance.

MATERIALS AND METHODS

Insects.

All bioassays were performed with a laboratory strain of S. nonagrioides established from field-collected larvae obtained from nontransgenic corn and reared on an artificial diet as described previously (7). Egg masses were obtained by confining a minimum of 100 pairs (batches of 5 to 10 pairs) of adults in ventilated plastic cylinders (diameter, 12 cm; height, 30 cm) containing five to seven corn seedlings for oviposition. Egg masses were removed and placed in plastic boxes containing moistened filter paper until hatching. The environmental conditions for larval rearing, mating, oviposition, and egg incubation were 25.0 ± 0.3°C, a relative humidity of 70% ± 5%, and a photoperiod consisting of 16 h of light and 8 h of darkness.

B. thuringiensis Cry proteins.

The following Cry proteins were produced in B. thuringiensis strains expressing just one type of Cry protein: EG1273 (Cry1Aa), EG7077 (Cry1Ab), EG11070 (Cry1Ac), EG19916 (Cry1Ba), EG1081 (Cry1Ca), EG7300 (Cry1Da), EG11096 (Cry1Fa), and EG7279 (Cry1Ja), obtained from Ecogen Inc. (Langhorne, Pa); EG7543 (Cry2Aa), obtained from Monsanto Co. (Chesterfield, Mo); and HD1-9 (Cry1Ab), obtained from Syngenta. For bioassays, crystal solubilization and protoxin activation (by treatment with bovine trypsin) were performed as described previously (21). Essentially, each B. thuringiensis strain was grown at 29°C for 48 h in CCY medium (32) supplemented with the appropriate antibiotic. Spores and crystals were collected by centrifugation at 9,700 × g at 4°C for 10 min. The pellets were washed four times with 1 M NaCl-10 mM EDTA and suspended in 10 mM KCl. Crystals were solubilized in 50 mM sodium carbonate buffer (pH 10.5) containing 10 mM dithiothreitol. Protoxins were trypsin activated at 37°C for 2 h (1 mg trypsin per 10 mg of protoxin). The protein concentrations in the preparations of Cry proteins were determined by the method of Bradford (4), using the Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Munich, Germany) with bovine serum albumin (BSA) as the standard.

Bioassays.

Neonate larvae (<24 h old) were confined individually in plastic trays (Bio-Ba-128; Color-Dec Italy, Capezzano Pianore, Italy) and exposed to surface-treated artificial diet as described previously (7). Tray wells filled with 1 ml of artificial diet, with an exposed surface area of 1.77 cm², were treated with 50-μl portions of different toxin dilutions; after drying in a laminar flow hood, a single neonate larva was confined in each tray by a plastic cover (Bio-Cv-16; Color-Dec Italy, Capezzano Pianore, Italy). To determine the appropriate concentration range, bioassays with three concentrations (10, 100, and 1,000 ng/cm²) and a negative control were performed with Cry1Aa, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Da, Cry1Fa, Cry1Ja, and Cry2Aa activated toxins to assess their toxicity for S. nonagrioides (referred to as “bracketing bioassays”). Toxins were diluted in 50 mM sodium carbonate buffer (pH 10.5), and the same buffer containing no toxin was used as a negative control. Each concentration was replicated twice, and each replicate included 16 observations (32 insects per concentration).

Bioassays to determine 50% lethal concentration (LC₅₀s) were performed only with the toxins that showed high levels of toxicity for S. nonagrioides in the bracketing bioassays and with Cry1Ab protoxin (crystals from strain HD1-9), whose toxicity was demonstrated previously (7). Cry1Ab crystals were resuspended in a 0.1% solution of Triton X-100, and serial dilutions in distilled water were prepared. Forty-eight larvae (three replicates of 16 larvae each) plus the control were tested for each concentration. Trays were incubated in growth chambers at 25.0 ± 0.3°C with a relative humidity of 70% ± 5% and constant darkness. Mortality (larvae that did not show any reaction when they were prodded were considered dead) was assessed after 7 days.

Probit analyses (9) using mortality data were performed with the computer program POLO-PC (LeOra Software, Berkeley, CA) to determine the susceptibility to the different toxins (LC₅₀s and 95% fiducial limits). The significance of differences in susceptibility was tested by determining the 95% fiducial limits of lethal concentration ratios (LCR) at the LC₅₀ (26).

Toxin purification and labeling.

Activated Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa proteins that were used in biochemical analyses were purified further by fast liquid protein chromatography (21). Cry1Ab, Cry1Ac, and Cry1Ca proteins were labeled with ¹²⁵I (Amersham, Little Chalfont, United Kingdom) using the chloramine T method (34); the specific activities of the labeled proteins were 5.11, 110, and 2.88 mCi/mg, respectively. Cry1Ab and Cry1Fa were labeled with biotin using the protein biotinylation module (RPN 2202; Amersham, Little Chalfont, United Kingdom) according to the manufacturer's protocol.

BBMV preparation and enzyme assays.

For pore formation assays, midguts were dissected from last-instar larvae, rinsed in a medium containing a mixture of protein inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 mM dithiothreitol, 1 μM pepstatin A, 2 μg/ml aprotinin, and 0.1 mM leupeptin), and preserved in liquid nitrogen. Brush border membrane vesicles (BBMV) were prepared from rapidly thawed midguts by Ca²⁺ precipitation and differential centrifugation as described previously (10). A short homogenization step (six strokes at 1,500 rpm in a glass-Teflon Potter) was necessary to obtain brush border fragments that were effectively closed to form tight vesicles.

The purity of a preparation was checked by evaluating the enrichment of the brush border membrane marker enzymes aminopeptidase N and alkaline phosphatase and of the basolateral membrane marker enzyme trehalase (1). Aminopeptidase N (EC 3.4.11.2) and alkaline phosphatase (EC 3.1.3.1) activities in the homogenate and in the BBMV preparation were determined as described previously (10). Trehalase (EC 3.2.1.28) activity was measured at 30°C by enzymatic colorimetric determination of the glucose released (Glucose Liquid; Sentinel, Switzerland) with a Pharmacia Biotech Ultrospec 3000 spectrophotometer.

For radioligand and biotin binding assays, BBMV were prepared by the differential magnesium precipitation method from dissected midguts of last-instar larvae (36). Protein concentrations in the BBMV preparations were determined by the method of Bradford, using BSA as the standard (4).

Binding assays with ¹²⁵I-labeled Cry proteins.

Prior to use, BBMV were centrifuged for 10 min at 16,000 × g and suspended in binding buffer (phosphate-buffered saline containing 0.1% BSA). Homologous and heterologous competition experiments were performed in binding buffer by incubating 20 μg BBMV with 67 pM ¹²⁵I-Cry1Ab, 3 pM ¹²⁵I-Cry1Ac, or 225 pM ¹²⁵I-Cry1Ca and different amounts of unlabeled toxin in 100-μl (final volume) mixtures for 1 h at 25°C. After incubation, samples were centrifuged at 16,000 × g for 10 min, and the pellets were washed twice with 500 μl of ice-cold binding buffer. The radioactivity in the pellets was measured with a model 1282 Compugamma CS gamma counter (LKB Pharmacia). For dissociation kinetics assays, labeled toxins were incubated with BBMV under the conditions described above before addition of a 1,000-fold excess of unlabeled toxin. Then we determined the time course of labeled toxin dissociation from BBMV. Equilibrium dissociation constants (K_d) and the concentrations of binding sites (R_t) were estimated using the LIGAND program (25).

Binding assays with biotinylated Cry proteins.

Biotinylated Cry1Ab and Cry1Fa were incubated for 1 h with 20 μg of BBMV in 100 μl of binding buffer. In competition experiments, excess unlabeled competitors were added before addition of the BBMV to the reaction mixtures. After incubation, samples were centrifuged at 16,000 × g for 10 min, and the pellets were washed with 500 μl of ice-cold binding buffer. The pellets were suspended in 10 μl of electrophoresis sample buffer (28). Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Hybond-ECL; Amersham Bioscience, Little Chalfont, United Kingdom). The membranes were incubated with a streptavidin-alkaline phosphatase conjugate (1:1,500; Roche Diagnostics GmbH, Mannheim, Germany). Biotinylated toxins were detected in the membrane by color development using nitroblue tetrazolium (NBT)-5-bromo-4-chloro-3-indolylphosphate (BCIP) (Roche Diagnostics GmbH, Mannheim, Germany) as the alkaline phosphatase substrate, as recommended by the manufacturer.

BBMV permeability assays.

BBMV were preloaded with 1 mM KCl by resuspending the membranes in 300 mM mannitol-1 mM KCl-10 mM HEPES-Tris (pH 7.2) before the last centrifugation of the Ca²⁺-based BBMV preparation procedure. Suitable amounts of BBMV were preincubated for 30 min at 4°C with the toxins at different concentrations (see the legend to Fig. 4).

FIG. 4.

Effects of different Cry proteins on K⁺ permeability in BBMV from S. nonagrioides. BBMV that were resuspended in 300 mM mannitol-1 mM KCl-10 mM HEPES-Tris (pH 7.2) were preincubated for 30 min with just the buffer in which the toxins were dissolved (control) or with Cry1Ab (15 μg/mg BBMV protein) (B), with Cry1Fa (25 μg/mg BBMV protein) (C), or with Cry1Da (40 μg/mg BBMV protein) (D). BBMV were then diluted in a spectofluorometer cuvette with the resuspension buffer supplemented with 6 μM DiSC₃(5). For panel A, 23 μM valinomycin was added to the diluting buffer. KCl was added at the times indicated by the arrows to obtain final concentrations of 20, 40, 60, and 80 mM. Each trace represents the means ± standard errors for different experiments performed six times (control) or three times. AU, arbitrary units.

The transmembrane electrical potential difference generated by extravesicular increments of 20 mM KCl was determined by using the voltage-sensitive dye 3,3′-diethylthiacarbocyanine iodide [DiSC₃(5)] (Molecular Probes, Socetà Italiana Chimici, Italy) with BBMV incubated with control and Cry proteins. Prior to use, Cry protein preparations were made Na⁺ free by dialyzing them overnight against 20 mM Tris-HCl-150 mM CsCl (pH 8.6) with regenerated cellulose SPECTRA/POR membranes (molecular mass cutoff, 12,000 to 14,000 Da; SPECTRUM U.S. & Canada). Fluorescence measurements were obtained by using a polyacryl cuvette with a spectrofluorometer (Jasco FP-777) equipped with a thermostatic (25°C) holder. The excitation and emission wavelengths were 645 and 665 nm, respectively (33). Fluorescence values were expressed in arbitrary units.

RESULTS

Susceptibility of S. nonagrioides to Cry proteins.

In the bracketing bioassays, five of the eight toxins tested, Cry1Aa, Cry1Ba, Cry1Da, Cry1Ja, and Cry2Aa, caused low levels of mortality of S. nonagrioides neonate larvae at the highest concentration tested (1,000 ng/cm²) (31%, 28%, 6%, 3%, and 63%, respectively). Only Cry1Ac, Cry1Ca, and Cry1Fa caused higher levels of mortality, which ranged from 88% to 97% at the highest concentration and from 9% to 50% at the 100-ng/cm² concentration. For all toxins, the lowest concentration assayed (10 ng/cm²) did not result in significant mortality (data not shown).

The toxicities of the Cry1Ab protoxin and of the three more active toxins selected based on the results of the bracketing assays were evaluated further by performing bioassays with different toxin concentrations in order to determine LC₅₀s. The lowest LC₅₀ observed was the LC₅₀ of Cry1Ab (23 ng/cm²), and the highest LC₅₀s were the LC₅₀s of Cry1Ca and Cry1Fa (190 and 297 ng/cm², respectively) (Table 1). The LCR were estimated by comparing the S. nonagrioides responses to different toxins with the response to the most toxic toxin (Cry1Ab). None of the confidence limits of the response ratios included the value 1.0 (Table 1), indicating that all the other LC₅₀s were significantly different from the LC₅₀ of Cry1Ab (P < 0.05). When the 95% confidence limits of the LC₅₀s were considered, they overlapped for Cry1Ac and Cry1Ca and for Cry1Ca and Cry1Fa. Hence, despite differences in absolute values, the toxicities of these toxins were very similar.

TABLE 1.

Results of probit analysis, indicating the susceptibility of a laboratory strain of S. nonagrioides to different Cry toxins

Toxin	n	Slope (mean ± SE)a	χ2	df	LC50 (ng protein/cm2 diet)	LC90 (ng protein/cm2 diet)	LCR (LC50)b
Cry1Abc	336	1.8 ± 0.2 ac	17.5	19	23 (16-30)d	119 (86-185)	1
Cry1Ac	329	1.3 ± 0.2 a	36.1	19	105 (47-175)	999 (575-2623)	4.6 (2.6-8.2)
Cry1Ca	283	2.7 ± 0.3 b	14.7	16	190 (163-220)	567 (458-766)	8.4 (5.8-12.2)
Cry1Fa	333	2.0 ± 0.2 bc	36.3	19	297 (182-435)	1,271 (834-2416)	13.1 (8.4-20.5)

^aValues followed by the same letter indicate that the probit lines are parallel when the slopes are constrained according to the POLO program (P < 0.05) (LeOra Software, 1987).

^bLC₅₀s are considered significantly different from the LC₅₀ of Cry1Ab (P < 0.05) if the LCR 95% confidence interval does not include 1.

^cThis Cry protein was tested as a crystalline inclusion (protoxin form).

^dThe values in parentheses are 95% confidence intervals.

Binding of ¹²⁵I-labeled Cry proteins to BBMV.

Binding assays performed with ¹²⁵I-labeled Cry proteins showed that Cry1Ab, Cry1Ac, and Cry1Ca bound specifically to the epithelial membrane of S. nonagrioides larvae (Fig. 1).

FIG. 1.

Binding of ¹²⁵I-Cry1Ab (A), ¹²⁵I-Cry1Ac (B), and ¹²⁵I-Cry1Ca (C) to BBMV from S. nonagrioides with different concentrations of unlabeled competitor. ▴ and solid line, Cry1Aa; • and dashed line, Cry1Ab; ▾ and dotted line, Cry1Ac; ⧫ and dashed and dotted line, Cry1Ca; ▪, Cry1Fa.

Quantitative analysis of the results of homologous competition assays (when the labeled and unlabeled toxins were the same) showed that the affinity of Cry1Ac for its binding sites was significantly greater than the affinity of Cry1Ab or Cry1Ca and that Cry1Ca was the toxin with the highest concentration of binding sites (about threefold) (Table 2).

TABLE 2.

Dissociation constants and concentrations of binding sites for binding of Cry proteins to BBMV from S. nonagrioides^a

Cry protein	Kd (nM) (mean ± SD)	Rt (pmol/mg) (mean ± SD)b
Cry1Ab	20 ± 9	1.8 ± 0.7c
Cry1Ac	4 ± 1	1.8 ± 0.7c
Cry1Ca	50 ± 8	5 ± 1

^aAll parameters were estimated from the results of homologous competition assays. The values are the means and standard deviations for at least two replicates.

^bExpressed as picomoles per milligram of total vesicle protein.

^cValue estimated from joint analysis of homologous competition data for labeled Cry1Ab and Cry1Ac. Since Cry1Ab completely competes for Cry1Ac binding sites and vice versa, we assumed that the concentration of binding sites is the same for both toxins.

In heterologous competition assays (when the labeled and the unlabeled toxins were different), we found that Cry1Ab and Cry1Ac completely competed with each other (Fig. 1A and B), indicating that all of their binding sites are shared. Cry1Aa also completely competed with ¹²⁵I-Cry1Ab and ¹²⁵I-Cry1Ac, indicating that it also bound to the common binding sites (Fig. 1A and B). However, assays performed with Cry1Fa showed that this toxin did not compete with either ¹²⁵I-Cry1Ab or ¹²⁵I-Cry1Ac. Similarly, Cry1Ac did not compete for ¹²⁵I-Cry1Ca binding sites (Fig. 1C). Thus, both Cry1Fa and Cry1Ca have their own independent high-affinity binding sites.

Since it is assumed that binding of Cry toxins includes an irreversible step to produce pores and exert its toxic action, we also determined the proportion of total binding that was irreversible. Dissociation kinetics assays showed that binding of ¹²⁵I-Cry1Ab and ¹²⁵I-Cry1Ac was very stable over time and that, in the time frame of the assay, it was completely irreversible (Fig. 2).

FIG. 2.

Dissociation kinetics of the binding of ¹²⁵I-Cry1Ab (• and dashed line) and ¹²⁵I-Cry1Ac (▾ and dotted line) to S. nonagrioides BBMV. After 1 h of incubation of the BBMV with the labeled toxins, the assay was initiated by adding an excess of unlabeled homologous competitor.

Binding of biotin-labeled Cry proteins to BBMV.

Since it is known that Cry1Fa cannot be labeled with ¹²⁵I without losing its biological activity (24), in order to perform reciprocal competition assays, we labeled this toxin with biotin and performed homologous and heterologous competition assays. Cry1Fa bound to BBMV (Fig. 3A, lane 1Fa*), and this binding was specific since an excess of unlabeled Cry1Fa decreased the signal intensity of the band (Fig. 3A, lane 1Fa). In addition, both Cry1Ab and Cry1Ac competed for Cry1Fa binding sites but apparently did so with very low affinity since there was only a slight reduction in the intensity of the band, compared with the almost complete absence of the band in the case of the homologous competition assay (Fig. 3A). As this result was unexpected based on the lack of competition of Cry1Fa obtained with ¹²⁵I-labeled Cry1A toxins, we decided to label Cry1Ab with biotin and perform the reciprocal experiment (Fig. 3B). Competition with Cry1Fa again resulted in a slight reduction in the intensity of the biotin-labeled Cry1Ab band (Fig. 3B, lanes 1Fa 2× and 1Fa 5×), which showed that Cry1Fa binds with very low affinity to the Cry1A binding site. The results also confirmed that Cry1Ab and Cry1Ac share a binding site and that the affinity of Cry1Ac is greater than that of Cry1Ab (based on the greater reduction in the intensity of the band when an excess of Cry1Ac was used than when an excess of Cry1Ab was used) (Fig. 3B, lanes 1Ab 2×, 1Ab 5×, 1Ac 2×, and 1Ac 5×).

FIG. 3.

Binding of biotin-labeled Cry1Fa (A) or Cry1Ab (B) to BBMV from S. nonagrioides in competition assays. (A) Lane MW, molecular weight markers (Precision Plus protein dual-color standard; Bio-Rad Laboratories GmbH, Munich, Germany); lane BBMV, BBMV without toxin; lane 1Fa*, BBMV with biotin-labeled Cry1Fa (Cry1Fa*); lane 1Fa, competition assay with Cry1Fa* and Cry1Fa (1,000×); lane 1Ab, competition assay with Cry1Fa* and Cry1Ab (1,000×); lane 1Ac, competition assay with Cry1Fa* and Cry1Ac (1,000×). (B) Lane MW, molecular weight markers; lane 1Ab*, BBMV with biotin-labeled Cry1Ab (Cry1Ab*); lane 1Ab 2×, competition assay with Cry1Ab* and Cry1Ab (2×); lane 1Ab 5×, competition assay with Cry1Ab* and Cry1Ab (5×); lane 1Ac 2×, competition assay with Cry1Ab* and Cry1Ac (2×); lane 1Ac 5×, competition assay with Cry1Ab* and Cry1Ac (5×); lane 1Fa 2×, competition assay with Cry1Ab* and Cry1Fa (2×); lane 1Fa 5×, competition assay with Cry1Ab* and Cry1Fa (5×); lane 1Ab*, BBMV with Cry1Ab*.

BBMV permeability assays.

To obtain a BBMV preparation that was relatively free of basolateral membranes, we used the Ca²⁺ precipitation method, which resulted in sevenfold increases in the specific activities of the two apical membrane enzymes and no increase in the trehalase specific activity (Table 3).

TABLE 3.

Protein contents and marker enzyme activities in homogenates and BBMV preparations from S. nonagrioides larval midguts^a

Parameter	Homogenateb	BBMVb	Enrichment factorc
Protein yield (mg/g fresh tissue)	36 ± 2	1.1 ± 0.2
Alkaline phosphatase activity (mU/mg protein)	160 ± 30	1,000 ± 160	6.9 ± 0.9
Aminopeptidase N activity (mU/mg protein)	250 ± 40	1,800 ± 300	7.3 ± 0.6
Trehalase activity (mU/mg protein)	70 ± 10	90 ± 17	1.3 ± 0.2

^aBBMV were prepared by the Ca²⁺ precipitation method.

^bThe values are means ± standard errors for 12 replicates for the protein yield and for five replicates for the enzyme activities.

^cRatio of the enzyme specific activity in BBMV to the enzyme specific activity in the homogenate.

The abilities of Cry1Ab and Cry1Fa, both of which are toxic to S. nonagrioides, and of Cry1Da, a nontoxic protein, to enhance the membrane permeability to K⁺ were assayed by recording the variation in the fluorescence of the voltage-sensitive dye DiSC₃(5) with increasing inwardly directed K⁺ gradients (Fig. 4). The fluorescence signals of the different BBMV preparations were normalized to the mean basal value recorded in the absence of the cation gradient. In control vesicles, progressive addition of KCl to obtain extravesicular concentrations of 20, 40, 60, and 80 mM generated transmembrane electrical potentials of different magnitudes with the positive pole inside the vesicles, which were recorded as increases in the fluorescence signal. When the membrane K⁺ permeability was enhanced by adding the specific K⁺ ionophore valinomycin (23 μM), higher diffusion potentials were obtained, and the fluorescence signal increased (Fig. 4A). In BBMV preincubated with a single dose of Cry1Ab or Cry1Fa (15 and 25 μg/mg BBMV proteins, respectively), a significant increase in the signal was observed, although the increase was considerably less than that induced by valinomycin. No evident increase in fluorescence was observed with 40 μg of Cry1Da/mg BBMV proteins (Fig. 4D). These results are in agreement with the in vivo activities of the Cry proteins tested.

DISCUSSION

Despite the fact that nearly 250 different B. thuringiensis toxins have been found (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/), it is known that only a few of these toxins are active against any given pest. Bt corn hybrids, which were developed primarily to control the European corn borer, Ostrinia nubilalis, have also exhibited good control of the Mediterranean corn borer (unpublished results). Laboratory bioassays with pure Cry1Ab protoxin to establish the baseline susceptibility of Spanish populations have indicated that S. nonagrioides is slightly more susceptible to this toxin than O. nubilalis is (12). In addition to Cry1Ab, other B. thuringiensis toxins, including Cry1Ac, Cry1Ba, Cry1Fa, Cry2Aa, and Cry9C, have been shown to be active against O. nubilalis (6, 17, 22, 30). However, this type of information was lacking for S. nonagrioides despite the fact that this insect is a major pest of corn in Mediterranean countries. In screening for other Cry proteins toxic to this pest, we selected some of the most common Cry proteins that are active against lepidopteran species. Our results show that in addition to Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Fa are toxic to S. nonagrioides. Although not as active as Cry1Ab, these toxins can be considered alternatives to Cry1Ab for control of this corn borer.

The generally accepted mechanism of action of Cry proteins includes a first reversible step that involves binding to specific receptors in the brush border membrane, followed by an irreversible step that is thought to reflect a conformational change that leads to insertion into the lipid bilayer (23). Insertion of several toxin molecules leads to the formation of lytic pores in the membrane (5, 31). Our data show that the four active toxins mentioned above bind specifically to the epithelial membrane of S. nonagrioides larvae. We have also found evidence of binding stability, and hence irreversibility, in two of these toxins (only Cry1Ab and Cry1Ac were tested). Furthermore, the variations in the fluorescence signal emitted by the voltage-sensitive dye DiSC₃(5) showed that Cry1Ab and Cry1Fa, both of which are active in vivo, were able to form pores that enhance the apical membrane permeability to K⁺. In contrast, Cry1Da, which is not toxic to larvae, did not show any pore formation activity.

Competition assays have shown that Cry1Aa, Cry1Ab, and Cry1Ac share binding sites and that Cry1Ca has its own nonshared binding site. This is also the general pattern for all insect species tested to date (8). Using reciprocal competition assays performed with both labeled Cry1Ab and labeled Cry1Ac, we determined that these toxins recognize the same binding sites. Although Cry1Aa was found to have poor toxicity, binding analysis showed that it also bound to the common site. However, as Cry1Aa was not labeled, we cannot exclude the possibility that this toxin could have binding sites other than the binding site shared with Cry1Ab and Cry1Ac. As in other cases, we did not find a direct correlation between the binding affinity and toxicity of Cry1Ab and Cry1Ac (35).

When Cry1Fa binding was examined, the lack of competition observed in the assays with ¹²⁵I-Cry1Ab and ¹²⁵I-Cry1Ac, along with its toxicity, suggested that Cry1Fa has its own independent binding sites. To show specific binding of this toxin, we labeled it with biotin and performed homologous competition assays with unlabeled Cry1Fa. Heterologous competition with unlabeled Cry1Ab and Cry1Ac, along with reciprocal experiments with biotin-labeled Cry1Ab, confirmed that Cry1Fa and the Cry1A proteins have different high-affinity binding sites. In addition, these experiments showed that there was slight cross-recognition of each other's binding sites by these toxins. Our results are very similar to those previously reported for O. nubilalis, for which different high-affinity binding sites for Cry1A proteins and Cry1Fa and slight cross-recognition between the proteins were also found (17). Thus, the patterns are the same for both corn borers, and they contrast with the patterns for other lepidopteran species, in which there is a common binding site for Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja, and Cry1Fa apparently does not have unique binding sites (13-15, 19).

A key requirement for the gene pyramiding strategy to delay the development of resistance is the absence of cross-resistance to the toxins expressed. In the absence of resistant populations, binding site analysis provides the basis of predictive models, and, in general, the results obtained have been shown to correlate well with the observed patterns of resistance to several Cry toxins (2, 11, 16, 20). Thus, in light of our binding model for S. nonagrioides we strongly recommend development of plants that express either Cry1Ab or Cry1Ac in combination with Cry1Ca or Cry1Fa. However, considering that the toxicity of Cry1Ac is significantly lower than the toxicity of Cry1Ab in S. nonagrioides and that Cry1Ca is not toxic to O. nubilalis (21, 22), the combination of Cry1Ab with Cry1Fa is more appropriate. Such plants would provide a better tool for resistance management of corn borer pest populations.