Occurrence of a common binding site in Mamestra brassicae, Phthorimaea operculella, and Spodoptera exigua for the insecticidal crystal proteins CryIA from Bacillus thuringiensis

Pergamon

PII: S0965-1748(97)00039-8

Insect Biochem. Molec. Biol. Vol. 27, No. 7, pp. 651–656, 1997  1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0965-1748/97 $17.00 + 0.00

Occurrence of a Common Binding Site in Mamestra brassicae, Phthorimaea operculella, and Spodoptera exigua for the Insecticidal Crystal Proteins CryIA from Bacillus thuringiensis BALTASAR ESCRICHE,† JUAN FERRE´,† FRANCISCO J. SILVA*† Received 5 December 1996; revised and accepted 6 May 1997

Specific binding to midgut membrane proteins is required for the toxicity of insecticidal crystal proteins (ICP) from Bacillus thuringiensis. A direct relationship between toxicity and binding has been proposed. It has been hypothesized that sharing of a single receptor by more than one ICP could lead to the occurrence of multiple resistance in the event of an alteration in the common receptor. Binding of CryIA(a), CryIA(b) and CryIA(c), three structurally related ICPs, has been studied in Phthorimaea operculella, Mamestra brassicae and Spodoptera exigua using brush border membrane vesicles (BBMV) from the midgut tissue. Using iodinated CryIA(b), the three insects showed similar results: one binding site for CryIA(b), which is shared with CryIA(a) and CryIA(c). The binding site concentrations obtained for CryIA(b) in P. operculella, M. brassicae and S. exigua were 5.1, 16.3 and 2.2 pmol/mg vesicle protein, respectively. In the same way, dissociation constants were 3.8, 5.3 and 0.7 nM. Data show that binding for an ICP does not directly imply toxicity. The occurrence of a common receptor for the CryIA subgroup of ICPs in P. operculella, M. brassicae and S. exigua might theoretically discourage the use of combinations of these ICPs in integrated pest management programmes.  1997 Elsevier Science Ltd Bacillus thuringiensis CryIA Phthorimaea operculella ceptor binding Insect resistance management

INTRODUCTION

New tactics in integrated pest management have focused on the application of environmentally friendly products as alternatives to chemical insecticides. Some of the most successful products are based on Bacillus thuringiensis. During sporulation, this Gram-positive bacterium produces proteinaceous crystalline inclusions with insecticidal properties. Each strain of B. thuringiensis produces a characteristic set of proteins, each toxic only to certain species of insects. These proteins are called insecticidal crystal

*Author for correspondence. Tel.: + 34 6 3983028; Fax: + 34 6 3983029; e-mail: [email protected] †Departament de Gene`tica, Universitat de Vale`ncia, C/Dr. Moliner 50, 46 100 Burjassot, Vale`ncia, Spain

Mamestra brassicae

Spodoptera exigua

Re-

proteins (ICP) or delta-endotoxins (Ho¨fte and Whiteley, 1989). Over 50 ICPs have been identified to date (Crickmore et al., 1995). All of them share some degree of sequence similarity, a feature which has been used to group them in different classes (CryI, CryII, CryIII, CryIV, etc.). For example, the proteins CryIA(a), CryIA(b) and CryIA(c) share an amino acid similarity of approx. 80% when the toxic fragments are compared (Ho¨fte and Whiteley, 1989). The ICP mode of action includes several steps: ingestion of the protein by the insect larva, solubilization and activation in its midgut, binding to specific receptors in the brush border of the midgut epithelium, and pore formation and disruption of the osmotic balance leading to the swelling and lysis of midgut cells with the death of the larva (Gill et al., 1992; Knowles, 1994). One of the most specific steps in the toxicity pathway

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is the binding of ICPs to midgut receptors (Hofmann et al., 1988a, b; Van Rie et al., 1989, 1990). Different situations have been found regarding the interaction between ICPs and binding sites. Several ICPs can thus have common binding sites, an ICP can bind to more than one site, or an ICP can bind to just a single site not recognized by other ICPs. Examples of the different situations were first reported by Hofmann et al. (1988b) and Van Rie et al. (1989) in Manduca sexta and Heliothis virescens. Resistance of insects to B. thuringiensis has been shown, in some cases, to be associated to reduced binding of ICPs to their receptors (Ferre´ et al., 1995). Resistance to Dipel (a commercial formulation containing CryIA(a), CryIA(b), CryIA(c), and CryII ICPs) in H. virescens has thus been explained by a modification in the common receptor for the three CryIA ICPs (Lee et al., 1995). Also, it has been suggested that cross-resistance to CryIF, in a strain of Plutella xylostella selected with Dipel (Tabashnik et al., 1994), is caused by a modification of a common receptor for CryIF and CryIA ICPs (Granero et al., 1996). Therefore, it seems to be important to determine the binding relationships between ICPs in target pests in order to avoid their combined use of those sharing a common receptor in integrated pest management systems. In the present work, we have examined the occurrence of binding sites for CryIA(b), and whether they are shared with other CryIA proteins (CryIA(a) and CryIA(c)) in three relevant insect pests: potato tuber moth (Phthorimaea operculella: Gelechiidae, Zeller), cabbage moth (Mamestra brassicae: Noctuidae, Linneo) and beet armyworm (Spodoptera exigua: Noctuidae, Hu¨bner). MATERIALS AND METHODS

Source of ICPs B. thuringiensis ICPs were obtained from Plant Genetic Systems (Ghent, Belgium). CryIA(a) and CryIA(b) were recombinant proteins expressed in Escherichia coli. CryIA(c) was isolated from B. thuringiensis var. kurstaki HD73. ICPs were supplied as activated trypsin-digested toxins (Van Rie et al., 1989). Insect rearing Insects were obtained from Plant Genetic Systems, and reared at 25°C and 60–70% relative humidity with a 16:8 h (light/dark) photoperiod. P. operculella was reared on potato tubers (Etzel, 1985), and last instar larvae were collected after emerging from the tubers before pupation. For M. brassicae and S. exigua artificial diets were used (David and Gardiner, 1966; and Patana, 1969, respectively). Binding experiments Experiments to determine the occurrence of binding sites for ICPs were performed according to Van Rie et

al. (1989). Iodination of ICPs was carried out using carrier-free [125I]-NaI (Amersham, Bucks., U.K.) and the oxidant agent chloramine-T. Evaluation of labelled toxin preparation was determined with sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS– PAGE) followed by gel autoradiography using X-Omat S film 100 (Kodak). Specific activity of iodinated ICPs was determined using a “sandwich” enzyme-linked immunosorbent assay (ELISA) technique, typically obtaining 0.5 mCi/mg. Iodinated ICP was used within a month after the date of labelling. Brush border membrane vesicles (BBMV) of M. brassicae and S. exigua were prepared from dissected larval midguts by the differential magnesium precipitation method described by Wolfersberger et al. (1987). A modified procedure was employed for P. operculella, which uses homogenized whole larvae as a starting material (Escriche et al., 1995). Protein contents of the BBMV preparations were determined by the method of Bradford (1976) by using bovine serum albumin (BSA) as the standard. Preliminary experiments were performed to determine the optimal assay conditions for binding of ICPs to BBMV for each insect species (Van Rie et al., 1989). Non-specific binding was determined in parallel experiments in the presence of 1 mM unlabelled protein. The value of non-specific binding was approx. 25% of total radioactive binding. The concentration of BBMV, incubation time, and labelled ICP concentration used in binding experiments were: 0.08 mg/ml of BBMV protein, 90 min incubation and 2.65 nM 125I-CryIA(b), for P. operculella; 0.11 mg/ml of BBMV protein, 90 min incubation, and 0.48 nM 125I-CryIA(b), for M. brassicae; 0.06 mg/ml of BBMV protein, 60 min incubation, and 0.44 nM 125I-CryIA(b), for S. exigua. All binding experiments were carried out at room temperature. Competition experiments were performed in the presence of different concentrations of non-labelled competitor in 0.1 ml of phosphate-buffered saline (PBS) 0.1% BSA buffer (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4, 0.1% BSA). Non-bound ICPs in samples were separated by filtration through Whatman GF/F glass fibre filters (Whatman Scientific Ltd, Maidstone, U.K.). The radioactivity retained in the filters was measured in a gamma-counter (Compugamma 1282, LKB). Data were analysed using the ligand computer program (Mundson and Rodbard, 1980), which calculates the bound concentration of ligand as a function of the total concentration of ligand, and gives estimates of the dissociation constant (Kd) (inversely correlated with the affinity) and the total binding site concentration (Rt). This computer program also compares one binding site vs two binding site models, testing if the increase in goodness of fit for the second model is significantly higher than would be expected on the basis of chance alone. The program uses an F-test based on the “extra sum of squares” principle.

COMMON RECEPTOR FOR CryIA

RESULTS

CryIA(b) has a single class of specific binding sites The occurrence of binding sites for CryIA(b) was determined by performing saturation experiments employing 125I-CryIA(b) and BBMV of P. operculella (Fig. 1(a)), M. brassicae (Fig. 1(b)) and S. exigua (Fig. 1(c)). The curves show the presence of specific receptors in the three cases with a maximum binding of 4% in P. operculella, 6% in M. brassicae and 23% in S. exigua. Homologous competition experiments using unlabelled CryIA(b) were done to determine the number of binding sites of the ICP in each insect and their binding parameters (dissociation constant, Kd, and receptor con(a)

centration, Rt). Results were similar in the three insect species (M. brassicae, P. operculella, and S. exigua) (Fig. 2). Data were fitted and compared with the ligand computer program, which gave better fitting to a one-site model than to a two-sites model (P ⬎ 0.05 in all cases). Binding parameters (Table 1) showed that the concentration of receptors was higher in M. brassicae and lower in S. exigua, with an intermediate value for P. operculella. Binding affinities of CryIA(b) (inverse of Kd) were higher for S. exigua and not significantly different for M. brassicae and P. operculella. CryIA(a), CryIA(b) and CryIA(c) proteins share a common binding site Heterologous competition experiments using unlabelled structurally related ICPs (CryIA(a) and CryIA(c)) I–CryIA(b)

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FIGURE 1. Specific binding of 125I-labelled-CryIA(b) to increasing amounts of BBMV of P. operculella (a), M. brassicae (b) and S. exigua (c). Values are the means and the standard deviations of two independent determinations. Non-specific binding was subtracted from the total binding for each data point. Specific binding was given as a percentage of total radioactivity assayed.

FIGURE 2. Binding of 125I-labelled-CryIA(b) as a function of increasing concentrations of unlabelled competitor to BBMV of P. operculella (a), M. brassicae (b) and S. exigua (c). Symbols correspond to CryIA(a) (䊊), CryIA(b) (䊐) and CryIA(c) (왕). Each point represents the mean and the standard deviation of duplicate samples.

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BALTASAR ESCRICHE et al. TABLE 1. Biochemical binding parameters of CryIA proteins

Insect species

M. brassicae S. exigua P. operculella

Rt

16.3 ± 2.9 2.2 ± 0.3 5.1 ± 0.9

Kd CryIA(a)

CryIA(b)

CryIA(c)

44 ± 14 1.5 ± 0.4 0.6 ± 0.3

5.3 ± 1.3 0.7 ± 0.1 3.8 ± 1.7

0.6 ± 0.2 1.2 ± 0.2 11 ± 2

Receptor concentration (Rt) is expressed as pmol/mg vesicle protein and dissociation constant (Kd) as nM.

were performed to test if the binding site for CryIA(b) was recognized by these other ICPs. In all cases, labelled CryIA(b) was displaced by unlabelled CryIA(a) and CryIA(c) (Fig. 2). However, the displacement efficiency of each ICP varied for each insect species (Fig. 2, Table 1). In M. brassicae CryIA(c) was the ICP with the highest affinity and CryIA(a) the one with the lowest, in P. operculella the situation was reversed, and in S. exigua the three ICPs had similar Kd values. DISCUSSION

In the present work we have demonstrated the occurrence of specific binding sites for CryIA(b) in the larval midgut of P. operculella, M. brassicae and S. exigua. Analysis of homologous competition data has rendered a single binding site model for CryIA(b) in the three insect species. Also, heterologous competition experiments showed that the CryIA(b) binding site is shared with CryIA(a) and CryIA(c) in these species. It is the first time that the binding site model for these three CryIA ICPs has been obtained for the species studied in this work. However, for S. exigua, interactions of these ICPs with denatured brush border proteins had already been reported (Oddou et al., 1993). The saturation curve in M. brassicae was clearly sigmoidal, in contrast to the hyperbolic shape of the other two. Both types of curves have been described in saturation experiments, although the sigmoidal ones are less frequent (Van Rie et al., 1990; Ferre´ et al., 1991; Cowles et al., 1995; Lee et al., 1995). To our knowledge, no explanation has ever been given for these alternative shapes, and they do not seem to affect further binding calculations. A direct relationship has been shown between biochemical binding parameters and the toxicity of ICPs in various insects species (i.e. M. sexta, H. virescens and Ostrinia nubilalis) (Van Rie et al., 1989, 1990; Denolf et al., 1993). However, cases where this relationship does not hold true have also been found (Wolfersberger, 1990; Ferre´ et al., 1991; Escriche et al., 1994). In the present work, because we did not perform toxicity tests, it is difficult to draw conclusions on this matter. However, using published data, we can compare toxicity with binding characteristics for CryIA(b) (because the other two ICPs were not labelled, we could have missed other binding sites not shared with CryIA(b)). It has thus been reported

that CryIA(b) is highly toxic to P. operculella (Van Rie et al., 1994), moderately toxic to M. brassicae (Ho¨fte and Whiteley, 1989), and that it has barely any toxic effect on S. exigua (Moar et al., 1990) The overall binding affinity of CryIA(b) for BBMV proteins of the three insect species, as measured by the Rt/Kd ratio, is lower for P. operculella (a value of 1.3) and equal for the other two species (a value of 3.1). Therefore, we do not find any relationship (direct or reversed) between reported toxicities and our binding results with CryIA ICPs in the insects studied. A practical aspect of characterizing binding sites for non-toxic (or negligibly toxic) ICPs, as in the case of CryIA ICPs in S. exigua, is that new recombinant ICPs could be designed with the binding domain of CryIA and other structural domains from different ICPs. This approach has recently been applied with success by constructing CryIA(b)-CryIC hybrids highly toxic to S. exigua (De Maagd et al., 1996). A common binding site for the three ICPs of the CryIA subgroup has been found in the present work for the three insects analysed, but had also been found in other lepidopteran species: in the tobacco hornworm (M. sexta: Sphingidae) (Van Rie et al., 1989), in the tobacco budworm (H. virescens: Noctuidae) (Van Rie et al., 1989), in the gypsy moth (L. dispar: Lymantridae) (Lee and Dean, 1996) and in the diamondback moth (P. xylostella: Plutellidae) (Ballester et al., 1994). The cabbage looper (Trichoplusia ni: Noctuidae) has a common binding site for CryIA(b) and CryIA(c), not shared by CryIA(a) (Estada and Ferre´, 1994). The European corn borer (O. nubilalis: Pyrilidae) (Denolf et al., 1993) has a common binding site for CryIA(b) and CryIA(c) (binding of CryIA(a) was not tested) and, also, the rice striped stem borer (Chilo suppressalis: Pyrilidae) (Fiuza et al., 1996) has a shared binding site for CryIA(a) and CryIA(c) (CryIA(b) was not tested). Since the above results include very taxonomically distant families of Lepidoptera, it seems to be a general feature of this order of insects that different CryIA ICPs share at least one common binding site, even for insect species that do not show susceptibility for these ICPs. Whereas in some cases insects have developed resistance to one or two CryIA ICPs while remaining susceptible to the rest (Ballester et al., 1994; Estada and Ferre´, 1994; McGaughey and Johnson, 1994), in others, insects

COMMON RECEPTOR FOR CryIA

have become highly resistant to CryIA(a), CryIA(b), and CryIA(c) (Tabashnik et al., 1993; Gould et al., 1995; Tang et al., 1996). In a resistant strain of P. xylostella, a single gene seems to confer resistance to the above three CryA ICPs and to CryIF (Tabashnik et al., 1997); since these four ICPs bind to the same receptor in P. xylostella, it is possible that this gene is involved in receptor formation. Cases of resistance to B. thuringiensis not due to receptor modification have also been described, however, the levels of resistance attained are much lower (Ferre´ et al., 1995). The three insects of the present study have a common binding site for CryIA(a), CryIA(b) and CryIA(c). In the case of M. brassicae and P. operculella, the use of B. thuringiensis formulations containing CryIA ICPs can, in the long term, select for resistance to the three of them (S. exigua is barely susceptible to CryIA ICPs and thus would not respond to selection). Therefore, to preserve the future use of this bioinsecticide, it is advisable that B. thuringiensis formulations include, in combination with CryIA ICPs, other ICPs binding to different target sites. P. operculella, for example, has independent binding sites for CryIA(b), CryIB, and CryIC (Escriche et al., 1994). REFERENCES Ballester V., Escriche B., Me´nsua J. L., Riethmacher G. W. and Ferre´ J. (1994) Lack of cross-resistance to other Bacillus thuringiensis crystal proteins in a population of Plutella xylostella highly resistant to CryIA(b). Biocontrol Sci. Tech. 4, 437–443. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248–254. Cowles E., Yunovitz H., Charles F.-J. and Gill S. S. (1995) Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut. Appl. Environ. Microbiol. 61, 2738–2744. Crickmore, N., Zeigler, D. R., Feitelson, J., Schnepf, E., Lereclus, D., Baum, J., Van Rie, J. and Dean, D.H., Bacillus thuringiensis deltaendotoxin nomenclature. WWW site: http://epunix.biols. susx.ac.uk/Home/Neil—Crickmore/Bt/index.html, 1995. David W. A. L. and Gardiner B. O. C. (1966) Rearing Mamestra brassicae (L) on semi-synthetic diets. Bull. Entomol. Res. 57, 137–142. De Maagd R. A., Kwa M. S. G., Van Der Klei K., Yamamoto T., Schipper B., Vlak J. M., Stiekema W. J. and Bosch D. (1996) Domain III in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Appl. Environ. Microbiol. 62, 1537– 1543. Denolf P., Jansens S., Peferoen M., Degheele D. and Van Rie J. (1993) Two different Bacillus thuringiensis delta-endotoxins receptors in the midgut brush border membrane of the European corn borer, Ostrinia nubilalis (Hu¨bner) (Lepidoptera: Pyralidae). Appl. Environ. Microbiol. 59, 1828–1837. Escriche B., Martı´nez-Ramı´rez A. C., Real M. D., Silva F. J. and Ferre´ J. (1994) Occurrence of three different binding sites for Bacillus thuringiensis ␦-endotoxins in the midgut brush border membrane of the potato tuber moth, Phthorimaea operculella (Zeller). Arch. Insect Biochem. Physiol. 26, 315–327. Escriche B., Silva F. J. and Ferre´ J. (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. Estada U. and Ferre´ J. (1994) Binding of insecticidal crystal proteins of Bacillus thuringiensis to the midgut brush border of the cabbage

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Acknowledgements—We are grateful to Plant Genetic Systems (Ghent, Belgium) for providing us with purified CryIA toxins. We also thank Barbara H. Knowles for critical comments on the manuscript. This work was supported by a grant from the European Union, under the ECLAIR program (project AGRE-0003), and a grant from the Ministerio de Agricultura, Pesca y Alimentacio´n (project AGR91-0238-CE). Support for B. Escriche was provided by a grant from the Ministerio de Educacio´n y Ciencia.