Determination of Receptor Binding Properties of Bacillus thuringiensis δ-Endotoxins to Cotton Bollworm (Helicoverpa zea) and Pink Bollworm (Pectinophora gossypiella) Midgut Brush Border Membrane Vesicles

Pesticide Biochemistry and Physiology 67, 198–216 (2000) doi:10.1006/pest.2000.2491, available online at http://www.idealibrary.com on

Determination of Receptor Binding Properties of Bacillus thuringiensis d-Endotoxins to Cotton Bollworm (Helicoverpa zea) and Pink Bollworm (Pectinophora gossypiella) Midgut Brush Border Membrane Vesicles Shahid Karim,*,1 S. Riazuddin,* F. Gould,† and Donald H. Dean‡ *National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan; †Department of Entomology, North Carolina State University, Raleigh, North Carolina 27695; and ‡Department of Biochemistry, Ohio State University, Columbus, Ohio 43210 Received August 13, 1999; accepted May 10, 2000 Pesticidal activity and receptor binding properties of Bacillus thuringiensis toxins to cotton pink bollworm (Pectinophora gossypiella) and cotton bollworm (Helicoverpa zea) were investigated. P. gossypiella was susceptible to Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa toxins. To H. zea, Cry1Ac and Cry1Ab were more potent than Cry1Aa and Cry2Aa. Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, Cry1Ha, and Cry2Ba were not potent against both pests. Binding assays were performed with 125I-labeled toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) and brush border membrane vesicles (BBMVs) prepared from H. zea and P. gossypiella midguts. Both Cry1Ab and Cry1Ac toxins showed saturable, high-affinity binding to P. gossypiella and H. zea BBMVs. Cry2Aa and Cry1Aa toxins bound to BBMVs with relatively low binding affinity but with high binding site concentration. Heterologous competition binding assays were performed to investigate the binding site cross reactivity. The results showed that Cry1Aa, Cry1Ab, and Cry1Ac recognize the same binding site, which is different from Cry2Aa. Ligand blot assay showed that Cry1Ac toxin binds to a 120kDa BBMV protein in P. gossypiella and Cry1Ab binds to a major 210-kDa protein. q 2000 Academic Press

INTRODUCTION

Bacillus thuringiensis (Bt) is a Gram-positive, aerobic spore former soil dwelling bacterium. Bt produces an enormous variety of intracellular proteinaceous crystalline inclusions known as dendotoxins after exponential growth is completed. The proteins comprising the crystal are the vanguard of active ingredients for biological control of commercially important insect pests, household pests, and vectors of animal and human diseases (1–3). Extensive screening programs have discovered different Bt strains with activity against species with seven different orders of insects, as well as against protozoa, flatworms, nematodes, and mites (4–6). Bt dendotoxin has been used successfully as a natural 1

To whom correspondence should be addressed at present address: 127 Noble Research Centre, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078. Fax: (405)-744-6039. E-mail: [email protected]. 198 0048-3575/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

pesticide in agriculture, forestry, and health for several decades due to the adverse effects of agrochemicals including lack of selectivity toward beneficial insects, environmental hazards, and health concerns as well as evolution of resistance. Resistance to chemical pesticides in more than 500 species of insects has been reported (7). Bt is widely applied for crops, forests, and environment protection. Each type of d-endotoxin is toxic to a limited range of insects. Based on their structural similarities and insect specificities, Bt toxins have been classified (8, 9). Recently, the nomenclature of Bt toxins has been revised on the basis of amino acid homology since protein structure is more relevant to Bt pesticidal action (10). In all cases of Bt pesticidal action documented to date, the target insect for toxicity must ingest the proteins. Therefore, Bt proteins are oral toxicants and have no demonstrable contact activity. Crystals are composed of protoxin proteins that

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are rendered toxic after they are solubilized under the alkaline conditions of the insect midgut (11). Lack of solubilization has been associated with the degree of toxicity (12, 13). After solubilization and proteolytic activation of the inclusions by sensitive larval midgut proteases, an active protease-resistant toxin core of molecular weight 55–70 kDa is formed. The activated toxin crosses the peritrophic membrane and binds to specific receptor molecules located in the microvillar brush border membranes (14– 17). After binding, the toxin irreversibly inserts into the membrane and alters the electrochemical potential gradient across the midgut by generating pores or selective/nonselective channels (18, 19). This destroys the osmotic balance of the cell membrane and causes the cell to swell and lyse. Recently, several common species of insect pests have evolved resistance to B. thuringiensis d-endotoxins (20–23). High levels of pest resistance have been reported in Plutella xylostella from the field selection (22, 24–26) and Plodia interpunctella, Heliothis virescens, Culex quinquefasciatus, Spodoptera exigua, Trichoplusia ni, and Leptinotarsa decemlineata in the laboratory selection (21, 27–32, 33–36). These reports support the results of laboratory selection for resistance to Bt in several pests (20, 21, 36). Researchers in industry, government, and academia now recognize the evolution of resistance to Bt in agronomically important pests as the greatest caveat to the continued success of Bt in the form of transgenic crop plants and transgenic bacteria (37–42). Elucidation of mechanisms of resistance of the insects to B. thuringiensis toxins could be critical for managing the rapid development of resistance. The mechanism of resistance could be related to disruption of the steps involved in the mode of B. thuringiensis toxin action such as ingestion, solubilization, activation of protoxin to toxin, crossing from peritrophic membrane, binding to the receptors, and pore formation (8, 11, 14, 15, 43, 44). The resistance is often related to a change in receptor binding properties on brush border membrane vesicles (BBMVs) of the insect midgut (16, 25, 30, 34, 45, 46). However, understanding the mechanism of resistance will help to develop strategies to delay the onset of resistance and

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hence prolong the usefulness of B. thuringiensis toxins as environmentally safe pesticides. Mixtures of toxin are an approach to delay and effectively control the pest population to more than one toxin simultaneously. This tactic is also called a multiple toxin approach, pyramid, or combination (39, 40, 47–49). The use of multiple toxins to impede evolution of resistance is based on the idea that if resistance to each component in a mixture is rare, then individuals with resistance to all components will be exceedingly rare or absent (50). The cotton pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) and cotton bollworm, Helicoverpa zea (Lepidoptera: noctuidae) are the most destructive pests of cotton worldwide. In Egypt, China, Brazil, and Pakistan, these pests commonly cause cotton losses of 20% or higher. The transgenic approach is the most promising technique for the control of both cotton pests. However, recent studies have demonstrated that interactions between toxins and binding sites may vary between toxins and target species (27). Thus, the development of transgenic cotton that is resistant to both pests will require identification of active B. thuringiensis toxins, as well as information on the interactions of the toxins with toxin binding proteins of midgut brush border membrane vesicles to develop cotton plants which produce a combination of different toxins which possibly delay the rapid onset of insect resistance and also control the insects effectively by acting synergistically. In the present study, we have investigated the toxicity of different Cry1A and Cry2A B. thuringiensis toxins to P. gossypiella and H. zea. We have also examined the receptor binding properties of potent toxins to the BBMVs of both pests and their relationship to the binding sites of each toxin. MATERIALS AND METHODS

Insects and Toxicity Assays Cotton pink bollworm (P. gossypiella) larvae and diet were the generous gifts of Dr. Fred Stewart (USDA, APHIS, PPQ, PBWRF, Phoenix, AZ). The bioassay with P. gossypiella was

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carried out with neonatal first-instar larvae on an artificial diet. Toxin was mixed thoroughly in the diet in food cups (purchased from Ohio State University, Food Facility) and dried in a laminar hood with small electric fans. Six different concentrations plus controls were tested with 30 larvae per cup. Each test was repeated four to five times. The mortality rates were recorded after 5 days of treatment and the effective dose estimates (50% lethal concentration of toxin (LC50) and 95% fiducial limits) were calculated using Probit analysis (51). Bioassays were conducted with neonate larvae of H. zea on modified artificial diet (52). A total of 100 ml (per well) of toxin dilutions (diluted in phosphate-buffered saline, 8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4) was layered on an artificial diet in a 24-well Falcon microtiter plate. One larva was placed per well (2 cm2) because of their cannibalism. The mortality rates were recorded after 5 days and the effective dose was estimated by Probit analysis. At least five concentrations per toxin were used to estimate the LC50 value. Each bioassay was repeated at least four times. Purification and Activation of Recombinant B. thuringiensis d-Endotoxins The cry1Aa, cry1Ab, and cry1Ac toxin genes wereover expressed in Escherichia coli JM103 by using the expression vector pKK223-3 (53). The cry2Aa was constructed in pTZ18R and expressed in E. coli (54). The Cry1Ba (DEC280pZ01220), Cry1Ca (DEC279-pSB607), Cry1Da (DEC314-pOH48), Cry1Ea (DEC293-pSB636), Cry1Fa (DEC 296), Cry1Ga (DEC284-pNZA10), and Cry1Ha were obtained from the Bacillus Genetic Stock Center (The Ohio State University, Columbus, OH). Bt d-endotoxins were purified from E. coli by a modification of the method of Hofte et al. (55). Inclusion bodies from Cry2Aa and other Cry1A’s were purified and solubilized as described previously (43, 52). The crystal protein was solubilized in alkalic buffer (50 mM sodium carbonate, 10 mM dithiothreitol, pH 9.5) for 2 h at 378C. The solubilized protoxin was digested with trypsin in a trypsin:protoxin ratio of 1:20 (by mass) for 2 h at

378C. Activated toxins of Cry1Aa, Cry1Ab, and Cry1Ac were dialyzed against 50 mM sodium carbonate buffer, pH 9.5. The solubilized protoxin of Cry2Aa was dialyzed against 50 mM sodium carbonate buffer, pH 10.5. The purity of protoxins and toxins was examined by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS–PAGE) (56). Protein concentrations of the protoxins and toxins were determined by the Bradford method (Pierce). The final concentration of the crystal protein used in the bioassay was estimated by scanning densitometery of gels stained with Coomassie brilliant blue (Sigma). Stability of d-Endotoxins in the Midgut Proteases The trypsin-activated Cry d-endotoxins were treated with P. gossypiella and H. zea midgut juice with a juice protein/activated toxin ratio of 1:1 (w/w) for different intervals of time at 378C. Gut juice of both target pests was obtained by squeezing the last-instar larvae from the abdomen. Gut juice digested toxins were analyzed by 10% SDS–PAGE. Proteins separated by SDS–PAGE were transferred to a PVDF membrane (Bio-Rad) using a Bio-Rad trans blot cell and detected by Cry1A and Cry2Aa polyclonal antibodies conjugated with IgG alkaline phosphate (Bio-Rad). Midgut Isolation and BBMVs Preparation Brush border membrane vesicles were prepared from isolated midguts of fourth-instar larvae of pink bollworm P. gossypiella and cotton bollworm H. zea. The larvae were placed on ice for 15 min; the head and abdomen were grasped with fine tweezers. Careful pulling separated the gut from the cuticle. The gut was placed in an ice-cold solution of Buffer A (MET-300 mM mannitol, 5 mM EGTA, 17 mM Tris–HCl, pH 7.5). Midguts were dissected from last-instar larvae and kept on liquid nitrogen until use. BBMVs were prepared by the differential magnesium precipitation method of Wolfersberger et al. (57). These BBMVs were resuspended in the buffer (8 mM NaHPO4, 2 mM KH2PO4, 150

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mM NaCl, pH 7.4) and the concentration of total protein was measured by Coomassie protein assay reagent (Pierce). Enzymatic assays were performed in triplicate on each homogenate and BBMV preparation. Alkaline phosphatase (EC3.1.3.1) activity was done on fresh samples as described (58). BBMVs from both preparations were examined by SDS–PAGE (10%). Iodination of Toxins The activated toxins were iodinated with Iodo-beads (Pierce) as specified by the manufacturer. A total of 1.0 mCi of Na 125I (Amersham) was added to a vial which contained one Iodobead (Pierce) and incubated for 5 min at room temperature. Toxin (50 mg) in 100 ml of 50 mM sodium carbonate buffer (pH 10.5) was added to the vial. After a 15-min incubation, the reaction was stopped by removing the Iodo-bead from the reaction vial. The reaction mixture was applied to a 2-ml Excellulose column (Pierce) that was equilibrated with 10 ml of 50 mM sodium carbonate buffer, pH 9.5, to remove free iodine and possible degradation products. The specific activity of the iodinated toxin was measured as follows. A total of 5 ml of labeled toxin was mixed with 100 ml of 20% trichloric acid and 100 ml of 2% BSA and incubated on ice for 10 min. The mixture was centrifuged in a Fisher microfuge at 13,500g for 10 min. The radioactivity of the pellet was counted in a gamma counter (Beckman). Another method to calculate the exact specific activity of the labeled toxin was also devised. The 2 ml of the labeled toxin was loaded on 10% SDS–PAGE. The resolved bands were cut from the gel and the radioactivity of the gel bands was counted in a gamma counter. The specific activities of labeled Cry toxins ranged from 0.6 to 1.2 mCi/mg protein. Binding Assays with BBMVs For the qualitative competition assay, 1 nM I-labeled toxin(1Aa, 1Ab, 1Ac, and 2Aa) was incubated with 10 mg of BBMV protein in the absence or presence of a 1000-fold excess of unlabeled toxins. After 1 h of incubation, reaction mixtures were separated by centrifugation 125

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at 13,500g for 10 min. The pellets were washed three times with binding buffer (8 mM Na2HPO4, 2 mM KH2PO4, and 150 mM NaCl (pH 7.4) containing 0.1% BSA) and separated by SDS– PAGE (12%). The dried gel was exposed to Fuji X-ray film for 1 to 3 days. For the quantitative binding assays, BBMVs were incubated with 125I-labeled toxins in 100 ml of binding buffer for 1 h at room temperature. Bound toxins were separated from unbound toxins by centrifugation at 13,500g for 10 min. The pellet containing the bound toxin was washed with binding buffer three times and the radioactivity of the resulting pellet was counted in a gamma counter (Beckman). Binding data were analyzed by using the Ligand computer program (59). Saturation binding assays were performed with fixed amounts of labeled toxins but varied amounts of BBMVs protein. 125I-labeled toxins (1 nM) were incubated with increasing BBMV concentrations (10 to 1000 mg). Nonspecific binding in the presence of excess amounts of unlabeled ligand (1000 nM) was subtracted from total binding for each datum point. In homologous and heterologous competition assays, BBMV (20, 10, 20, 10 mg for 1Aa, 1Ab, 1Ac, and 2A, respectively) were incubated with 1 nM labeled 1Aa, 1Ab, 1Ac, and 2Aa toxins for 1 h in the presence of increasing amounts of unlabeled competitors (0.25 to 1000 nM). Identification of d-Endotoxin Binding Proteins by Ligand Blotting Brush border membrane vesicles from the midgut of both target pests were solubilized in 1% Chaps and separated on 7.5% SDS–PAGE and transferred onto a PVDF membrane on transblot cell of Bio-Rad following the manufacturer’s instructions using transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). The membrane incubated for 2 h at room temperature in TTBS containing 2.5% BSA to block nonspecific binding. After being blocked, 125I-labeled Cry1Ab and Cry1Ab toxins and (10 nM) were incubated with membrane for 3 h at room temperature. After extensive washings, the membrane was exposed to X-ray film for 5 days.

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Autoradiography 125

I-labeled proteins were incubated with P. gossypiella and H. zea BBMV for 1 h at room temperature. The samples were centrifuged for 10 min at 13,500g in a Fisher microcentrifuge. The pellet was resuspended in binding assay buffer and centrifuged. The final pellet containing bound toxins and the supernatant containing free toxins were separated on SDS–PAGE (12%) (54). The dried gel was exposed to Fuji RX film for 24 h. Determination of Protein Concentration Protein concentrations of purified toxins and BBMVs were measured as described (60). RESULTS

Bt d-endotoxin proteins Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, Cry1Ha, Cry2Aa, and Cry2Ba were evaluated in a multiple-dilution bioassay against the most destructive pests of cotton, P. gossypiella and H. zea (Tables 1 and 2). The LC50 for Cry1Aa was 148.85 ng/mg of diet; Cry1Ab, 116.42 ng/mg of diet; and Cry1Ac, 66.15 ng/mg of diet whereas the LC50 of Cry2A was 153.537 ng/mg of diet. Cry1Ac toxin (LC50: 39.33 ng/cm2) was approximately 10 times more active than Cry1Aa (LC50: 382.61ng/cm2) and 13 times more than Cry2Aa (514.97 ng/cm2). Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, and Cry1Ha did not give any activity up to 400 ng/mg of diet. The purity of all Cry proteins was checked on 10% SDS–PAGE. Cry2Aa and other activated

Cry1A were quite stable in the gut juice of both target pests, namely P. gossypiella and H. zea (data not shown). Cry1A protoxin rapidly diminished as it was converted to activate toxin, but the activated 65-kDa Cry1A demonstrated no significant proteolytic loss during different time intervals. These data suggested that there was not a significant difference in the stability of all toxins so each may have an equal opportunity to bind to the midgut brush border. The purity of the brush border membrane vesicles prepared from the H. zea and P. gossypiella midguts was assessed by the distribution of alkaline phosphatase marker enzyme activity. The specific activity of brush border membrane marker enzymes, alkaline phosphatase, and leucine aminopeptidase was increased approximately 10 and 8 times, respectively, in the BBMV preparation relative to the midgut homogenate. The 125I-labeled Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa toxins showed a single 65-kDa band on SDS–PAGE (data not shown). Labeled toxins were incubated with BBMV from P. gossypiella and H. zea for 1 h, and the bound and free toxins were analyzed by SDS–PAGE and autoradiography. We observed that all 125I-labeled toxins were stable in P. gossypiella and H. zea BBMVs in 1-h incubations (Figs. 1A and 1B). The saturation binding assays were performed with a fixed amount of labeled toxins, but varied amounts of vesicle protein of P. gossypiella (Fig. 2). 125I-labeled toxins were incubated with increasing amounts of BBMVs (25 to 600 mg/ ml). Nonspecific binding in the presence of excess amounts of unlabeled ligand (mM) was

TABLE 1 Biotoxicity and Binding Kinetics of Purified d-Endotoxins of Bacillus thuringiensis against the First-Instar Larvae of Cotton Pink Bollworm (Pectinophora gossypiella) Toxin Cry1Aa Cry1Ab Cry1Ac Cry2Aa

LC50 ng/mg of diet (95% fiducial limit) 148.857 116.42 66.151 153.537

(85.256–428.078) (85.16–165.45) (29.76–145.04) (93.559–280.839)

Bmax (pm/mg of BBMV) 1.79 0.024 0.012 2025

6 6 6 6

0.01 0.013 0.015 2.130

Kcom (nM) 26.86 0.013 0.071 117.0

Note. Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, Cry1Ha, and Cry2Ba .400 ng/mg LC50 value.

6 6 6 6

0.102 0.011 0.019 3.21

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TABLE 2 Biotoxicity and Binding Kinetics of Purified d-Endotoxins of Bacillus thuringiensis against the First-Instar Larvae of Cotton Bollworm (Helicoverpa zea) Toxin Cry1Aa Cry1Ab Cry1Ac Cry2A

LC50 ng/cm2 of diet (95% fiducial limit) 382.61 73.68 39.33 514.97

(220.96–590.11) (51.83–113.72) (28.24–60.39) (363.57–659.13)

Bmax (pm/mg of BBMV) 1.26 0.017 5.751 187.50

6 6 6 6

0.11 0.023 0.401 2.913

Kcom (nM) 20.47 0.665 5.09 3.96

6 6 6 6

1.102 0.119 0.219 1.231

Note. Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, Cry1Ha, and Cry2Ba .1000 ng/cm2 LC50 value.

subtracted from the total binding for each data point. Labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins bound to BBMVs saturably at the concentration of 200 mg of vesicle protein/ml. Cry2Aa bound nonsaturably to the P. gossypiella brush border. H. zea brush border membrane vesicles demonstrated saturable binding to Cry1Aa, Cry1Ab, and Cry1Ac similar to that observed on brush border membrane vesicles from other Lepidoptera. Unlike Cry1A’s, Cry2Aa bound nonsaturably to H. zea brush border membrane vesicles (Fig. 3). These experiments demonstrated a correlation between toxicity and binding. Homologous competition studies (competition between labeled and unlabeled ligand) for labeled Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A were performed on P. gossypiella and H. zea

BBMVs (Figs. 4A–4D). From these experiments, we calculated the binding affinity (Kcom) and binding site concentration (Bmax) by using the Ligand computer programm (Tables 1 and 2). Cry1Aa toxin shows a single low-affinity site with an equilibrium constant, Kcom, of 26.86 nM, and a binding site concentration of 1.79 pmol/ mg of vesicle protein of P. gossypiella. Cry1Ab and Cry1Ac, which are more toxic to P. gossypiella, bound with high affinity to the vesicles (Table 1). Cry2Aa bound to the BBMVs with low affinity with an equilibrium constant, Kcom, of 117.00 nM and a binding site concentration of 2025 pmol/mg of vesicle proteins (Table 1). Homologous competition studies for labeled Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa were undertaken on H. zea BBMVs (Figs. 5A–5D)

FIG. 1. (A) Determination of stability and binding of 125I-labeled Cry toxins after incubation with P. gossypiella and (B) H. zea BBMV, lane 1, Cry1Aa; lane 2, Cry1Ab; lane 3, Cry1Ac; and lane 4, Cry2A.

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FIG. 2. Saturation binding of 125I-labeled Cry toxins to P. gossypiella BBMVs. (A) Saturation curve of Cry1Aa-labeled toxin, (B) Cry1Ab-labeled toxin, (C) Cry1Ac-labeled toxin, and (D) Cry2A-labeled toxin.

also. Cry1Aa and Cry2Aa toxins show a lowaffinity binding site with an equilibrium dissociation constant, Kcom, of 26.86 nM and 117.0 nM, respectively, and a binding site concentration of 1.79 and 2025 pmol/mg of vesicle protein. Both Cry1Ab and Cry1Ac toxins bound with a high affinity to the vesicles (Table 2). Our data gave further evidence that toxicity is correlated with high-affinity binding. Heterologous competition studies (competition between a labeled toxin and another unlabeled toxin) were performed with labeled and unlabeled Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa on both target pests, P. gossypiella and H. zea (Figs. 4 and 5). Labeled Cry1Aa is competing with unlabeled Cry1Ab and Cry1Ac in

both insects, whereas there is no competition between Cry1Aa and Cry2Aa for any binding site. Labeled Cry1Ab also shares some binding sites with Cry1Ac and Cry1Ab and did not share any site with Cry2Aa. Labeled Cry1Ac did not share any binding site with unlabeled Cry1Aa, Cry1Ab, and Cry2Aa in H. zea. Labeled Cry1Ac do share a few binding sites with Cry1Aa and Cry1Ab in P. gossypiella, whereas it did not compete with Cry2Aa. Labeled Cry2Aa toxin did not compete for binding of unlabeled Cry1Aa, Cry1Ab, and Cry2Aa in both target pests. In P. gossypiella, Cry1Ab and Cry1Ac toxins bound to major BBMV proteins of about 210 and 120 kDa (Fig. 6). In H. zea, Cry1Ab and

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FIG. 2—Continued

Cry1Ac bound to 170 and 150, 140, and 120 kDa. Our results with H. zea are in agreement with previous results (61). DISCUSSION

In present study, the relative activity of single proteins, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ga, Cry1Ha, Cry2Aa, and Cry2Ba, was compared. The ratio of differential activities of all purified d-endotoxins proteins varied. This difference is of particular importance in choosing suitable toxins. The supreme motivation for studying pesticidal activity of d-endotoxins is to better understand the molecular basis of their target

insect specificity as well as to assess their potential use in insect control programs. Of particular interest are the structural factors that enable toxins to bind receptors in the brush border membrane permeability, processes which are currently believed to be major determinants of specificity (15, 62, 63). Other factors that can play a key role include rates of ingestion and gut clearing (63), solubilization of inclusion proteins (12, 64), protoxin stability (65), proteolytic activation (66), and stability of the activated toxins in the larval midgut (67). The interpretation of published specificity data is often confounded by these factors, especially when diet-contamination methods are used to administer the toxins.

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FIG. 3. Saturation binding of 125I-labeled Cry toxins to H. zea BBMVs. (A) Saturation curve of Cry1Aa-labeled toxin, (B) Cry1Ab-labeled toxin, (C) Cry1Ac-labeled toxin, and (D) Cry2A-labeled toxin.

Our experiments established the toxicity of different Bt toxins in the order of Cry1Ac.Cry1Ab.Cry1Aa.Cry2Aa. Cry1 against P. gossypiella and H. zea. We also observed that labeled toxins were stable in the presence of BBMVs for 1 h (Fig. 1). Recent advances in the recombinant DNA technology had raised a question about the safety of transforming B. thuringiensis toxin genes into plants, which has been addressed (68). This question arises because truncated genes expressing the activated toxin are normally transformed into plants (the whole gene results in plant necrosis for unknown reasons), and concern was expressed as to the ability of the activated toxin interacting with mammalian gut tissue (68). B. thuringiensis

truncated cry genes have been transformed and successfully expressed in a wide variety of crops, including tomatoes, potatoes, cotton, maize, and rice (68). A field study of transgenic cotton Gossypium hirsutum L. expressing modified B. thuringiensis var. kurstaki protoxin in Arizona revealed high levels of resistance to the pink bollworm P. gossypiella (Saunders), even though the larvae initially penetrated the bolls (69). The saturation binding data on the BBMVs from P. gossypiella and H. zea demonstrated that Cry1Ac, which is the most potent toxin to both target pests larvae, bound to BBMV saturably with high affinity. Surprisingly, Cry1Aa, which is less toxic to P. gossypiella and H. zea, also

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FIG. 3—Continued

showed saturable binding with low affinity and lower binding site concentration than Cry1Ac protein. Cry2Aa, which is active against both pests, did not bind saturably. Cry1Ab exhibited specific and saturable binding with high affinity. These saturation binding data correspond to the bioassay to P. gossypiella. In both pests, direct correlation between toxicity and high-affinity receptor binding was observed; most potent toxins Cry1Ac and Cry1Ab showed high-affinity saturable binding, except Cry1Ac in H. zea showed low affinity. Cry1Aa, which is less active as Cry1Ac toxins to both pests, did show saturable binding. Furthermore, Cry2Aa toxin, which shows a little toxicity but is not as toxic as other Cry1A toxins to P. gossypiella and H.

zea, did not show saturable binding. Homologous competition assays were also performed with labeled Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa toxins to both P. gossypiella and H. zea (Figs. 4 and 5). Toward P. gossypiella and H. zea BBMV, even though Cry1Aa is less toxic to these insects, Cry1Aa, Cry1Ab, and Cry1Ac toxins bound to the membrane specifically, saturably, and with binding affinities. On the other hand, Cry2Aa, which is less potent than Cry1Ac toxin to both insects, showed competition with relatively lower binding affinities than Cry1Ac. These homologous competition data are in good agreement with the saturation binding assay data. The binding parameters, binding affinities

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FIG. 4. Homologous and heterologous competition binding assay of labeled Cry toxins to P. gossypiella BBMV. Binding of the labeled Cry toxins in the presence of increasing concentrations of unlabeled Cry toxins. (A) competition binding with labeled Cry1Aa competitor, (B) competition binding with labeled Cry1Ab, (C) competition binding with labeled Cry1Ac and unlabeled Cry toxins, and (D) competition binding with labeled Cry2A and cold Cry toxins.

(Kcom), and binding site concentration (Bmax) of various toxins to P. gossypiella and H. zea midgut were calculated (Tables 1 and 2). The binding affinities of Cry1Ac and Cry1Ab were comparable against both pests, at 5.09 nM. The binding of Cry1Ac to H. zea requires special remarks. Cry1Ac, which is the most potent toxin to H. zea, showed low affinity. This discrepancy could be explained in two ways. Even though binding affinities of Cry1Ac are lower than for the Cry1Ab toxin, the binding site concentrations are also lower. It has already been observed that the difference in insecticidal spectrum toward H. virescens is not due to the binding affinity

but to the difference in binding site concentration (43). Second, it appears that mechanism of action of B. thuringiensis protein is a two-step process; initial binding of Bt to the specific receptor is followed by membrane disruption by integration of the protein to the membrane. This would mean that binding to the receptor only partly determines the toxicity. Therefore, it could be possible that a postbinding event such as integration of protein into the membrane or efficiency of pore formation may be more important than the initial binding. Presumably, these binding affinities could be high enough to bind to the membrane receptor tightly and integrate into

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FIG. 4—Continued

the membrane and form a pore with about the same efficiency as in other pests. Recent developments in recombinant DNA technology offer the promise of the even greater use of Bt toxins in genetically engineered pestresistant crops. However, the fact that insects are able to develop resistance to B. thuringiensis toxins is an essential determinant for the longterm usefulness of this potential biocontrol agent. Resistance to insecticides is a major agricultural and public health concern. Resistance to pesticides in more than 500 species of insects (7) has taught us not to underestimate the evolutionary potential of pests (48). Eight species have been selected for resistance to B. thuringiensis d-endotoxins (46). The Diamond back moth, Plu. xylostella, is noteworthy as the

only pest having definitely evolved high levels of resistance in the field as a result of repeated use of B. thuringiensis (70). In laboratory selection experiments, high levels of resistance were obtained in the Indian meal moth (Plo. interpunctella) (70, 71). Resistance in Cadra cautella, H. virescens, and the Colorado potato beetle, L. decemlineata, also has been reported only from laboratory selection experiments (46). Resistance levels in two mosquito species (Aedes aegypti, Cu. quinquefasciatus) were relatively low (73, 74). In the field, Plu. xylostella evolved resistance against HD-1 (B. thuringiensis subspecies kurstaki) and, interestingly, HD-1 composed of 13.6% Cry1Aa, 54.2% Cry1Ab, and 32.2% Cry1Ac (75). In Plo. interpunctella, Plu. xylostella, and H. virescens, the resistance is due

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FIG. 5. Homologous and heterologous competition binding assay of labeled Cry toxins to H. zea BBMV. Binding of the labeled Cry toxins in the presence of increasing concentrations of unlabeled Cry toxins. (A) competition binding with labeled Cry1Aa competitor, (B) competition binding with labeled Cry1Ab, (C) competition binding with labeled Cry1Ac and unlabeled Cry toxins, and (D) competition binding with labeled Cry2A and cold Cry toxins.

to a change in binding affinity of receptors and binding sites on the BBMVs of the insect midgut (16, 25, 31). It may be a possibility that resistance is relatively specific to the toxin used in selection. This specificity has led to the possibility that resistance can be managed with the use of mixtures or sequences of unrelated toxins. However, evidence that cross resistance among toxins occurs in H. virescens was observed (28). Indeed, a strain of H. virescens selected for resistance to Cry1Ac was cross resistant to Cry1Aa, Cry1Ab, Cry1Ba, Cry1Ca, and Cry2Aa toxins. These findings provide little encouragement for the multiple-toxin approach to the resistance

management strategy in H. virescens. However, there are well-documented cases where insects have developed resistance to a certain crystal protein by changing their receptor for the Bt toxins, while being fully susceptible to other crystal proteins (17, 25, 76). The multiple-target strategy exploits the fact that while, in a population, insects homozygous for one resistance gene are rare, insects homozygous for multiple resistance genes are extremely rare. When using multiple crystal proteins, even insects homozygous for one or two resistance genes but heterozygous for another resistance gene would still be controlled by crops expressing multiple Bt toxins.

RECEPTOR BINDING PROPERTIES OF B. thuringiensis

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FIG. 5—Continued

The prime condition is very low cross resistance for the different crystal proteins. Therefore, heterologous competition assays were performed to investigate the cross reactivity of toxins and receptor binding sites. The binding data showed that Cry1Ac protein competed for the binding of labeled Cry1Ab and Cry1Aa with high affinity to P. gossypiella and H. zea (Figs. 4 and 5). Also Cry1Aa and Cry1Ab toxin competed for the binding of labeled Cry1Ac toxin on these insects (Figs. 4 and 5). These heterologous binding data demonstrated that Cry1Aa, Cry1Ab, and Cry1Ac recognize the same or common binding sites on P. gossypiella and H. zea midgut epithelial membrane. The finding of cross reactivity in binding sites for

Cy1Aa, Cry1Ab, and Cry1Ac toxins is not surprising, because experiments with H. virescens BBMV (30) also demonstrated that these two toxins share a common binding site. To P. gossypiella and H. zea, Cry2Aa toxin competed for the binding of labeled Cry1Aa and Cry1Ab toxins only marginally at high concentrations of the competitor (Figs. 4D and 5D). Cry1Ac toxin showed partial competition for the binding of Cry2Aa only at high concentrations of competitor on both insects (Figs. 4D and 5D.). Cry2Aa has a higher binding site concentration than Cry1Aa, Cry1Ab, or Cry1Ac on P. gossypiella and H. zea. The Cry2Aa binding results require special comment. The recent studies on the mode of action of Cry2Aa in H. zea (77) indicate that

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B

FIG. 6. Binding of 125I-labeled Cry toxins to protein blots of (A) P. gossypiella (lane 1, Cry1Ac; and lane 2, Cry1Ab) and (B) H. zea BBMVs (lane 1, Cry1Ab; and lane 2, Cry1Ac).

Cry2Aa does not show saturable binding to BBMVs and it did not inhibit subsequent binding of labeled Cry1Ac. In contrast, cold Cry1Ac inhibited the nonsaturable binding of Cry2Aa. Additionally, Cry2Aa did not lead to the formation of voltage-independent cation -selective channels in planar lipid bilayers, a characteristic

of Cry1Ac. They had suggested a unique mode of action of Cry2Aa. In contrast, our data showed that cold Cry1Ac did not displace all of the labeled Cry2Aa toxin from the BBMV, which suggested that a common binding site is being shared by both toxins at high concentrations. Cry1Ac toxin showed only partial competition

RECEPTOR BINDING PROPERTIES OF B. thuringiensis

for the binding of labeled Cry2Aa. These binding data demonstrated that Cry1Aa–Cry2Aa and Cry1Ab–Cry2Aa did not recognize the same binding site as Cry1Aa–Cry1Ab toxins. Ligand blot data showed that Cry1Ab binds to a major 210-kDa BBMV protein in P. gossypiella and a 170-kDa toxin binding protein in H. zea (Fig. 6). In the case of Cry1Ac the toxin binds to a major 120-kDa BBMV protein in P. gossypiella and different molecular weight BBMV proteins ranging from 150 to 100 kDa (155, 140, and 120 kDa) in H. zea. Our ligand blot data of H. zea is in agreement with previously published results (61). In conclusion, Cry1Aa, Cry1Ab, and Cry1Ac are not good choices for controlling cotton bollworms in transgenic plants because of their cross reactivity in receptor binding. Cry2Aa might be used in combination with the Cry1A type of toxins, due to their lack of cross reactivity. Cry2Aa toxin, which has a very different amino acid sequence from that of Cry1Ac, could be used with a combination of Cry1Ac toxin because of possible differences in the mode of action (77). Therefore, on the basis of these results, a future strategy could be devised for comparing Bt deployment systems in transgenic cotton in containment facilities to control pink bollworm and cotton bollworm; Cry1Ac alone; Cry1Ab alone; Cry2Aa alone; Cry1Ab or Cry2Aa together; and Cry1Ac or Cry2Aa together. A multitoxin deployment system will enhance the efficacy of the toxins and eventually slow the rate of adaptation of cotton bollworms to B. thuringiensis. Thorough research is needed to elucidate the patterns of different insect species before suitable recommendation is made which assures prevention of resistance.

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ACKNOWLEDGMENTS We thank Daniel R. Zeigler and Mi K. Lee for their critical review of the manuscript. S.K. has a fellowship from the Rockefeller Foundation. This work was supported by Grant R01 A1 29092 from the NIH to D.H.D.

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