Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera)

Pesticide Biochemistry and Physiology 85 (2006) 104–109 www.elsevier.com/locate/ypest

Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera) Shudong Luo a,b, Guirong Wang a, Gemei Liang a, Kong Ming Wu a,¤, Lianyang Bai b, Xinguo Ren b, Yuyuan Guo a a

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, PR China b College of Bio-safety Science and Technology, Hunan Agricultural University, Changsha 410128, PR China Received 24 January 2005; accepted 11 November 2005 Available online 10 January 2006

Abstract Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three 125I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no signiWcant diVerence in Cry1Ac-binding aYnity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (Rt decreased from 5.87 § 1.40 to 2.23 § 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance Wtted the “mode 1” pattern of Bt resistance described previously. © 2005 Elsevier Inc. All rights reserved. Keywords: Helicoverpa armigera; Cry1A toxins; Binding site; Binding model; Resistance

1. Introduction Bacillus thuringiensis (Bt), the most widely used biological insecticide, produces many kinds of insecticidal toxins during its sporulation [1,2]. These toxins are highly toxic to some insects, and harmless to most other organisms, including humans and other beneWcial insects. Each type of toxin has a unique spectrum of activity and targets. Among them, Cry1A is selective to lepidopteran larvae [3]. The action of B. thuringiensis crystal inclusions in insects is complex. Upon digestion by susceptible insect larvae, the inclusion bodies are solubilized, and the protoxins are converted to toxins. The activated toxins bind to receptors on the surface *

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of mid-gut epithelial cells of susceptible insects, and result in the lysis of the mid-gut epithelial cells and death of the insect [4–7]. As one of the most important biotechnological advances, Cry1Ac-transgenic cotton is being increasingly cultivated on a large scale. In 1997, Cry1Ac-transgenic cotton was Wrst planted in China, and it has spread over more than 2 million ha in 2003, which helped greatly to control Helicoverpa armigera [8,9]. Evolution of resistance by pests is the greatest threat to the continuous success of the Bt toxins used in conventional sprays or transgenic plants [2,10–12]. So far, only the diamondback moth, Plutella xylostella, has evolved resistance to Bt in the Weld. However, many pests, including H. armigera, have shown the capacity to develop resistance to Bt toxins in the laboratory screen tests [7,10,13–19]. To maintain the eVectiveness of Bt cotton, it is necessary

S. Luo et al. / Pesticide Biochemistry and Physiology 85 (2006) 104–109

to understand clearly the mechanisms of pest resistance to Bt toxins. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut [17,20,21]. Here, we report the research results on toxin binding in resistant and susceptible strains of H. armigera, and the binding models between the brush border membrane vesicles (BBMV) of H. armigera and Cry1A proteins. 2. Materials and methods

1

2

3

4

105

5

6

7

8

9

10

Fig. 1. Autoradiograph of 125I-labeled Cry1A toxins. Lanes: 1–4, 125 I-Cry1Aa; 5–7, 125I-Cry1Ab; 8–10, 125I-Cry1Ac.

2.1. Insects A Cry1Ac-susceptible strain of H. armigera was collected from Xinxiang, Henan Province, China, in 1996 and has been cultured for several years in the laboratory on artiWcial diet without Bt toxin. The Cry1Ac-resistant strain derived from the susceptible strain was selected using Cry1Ac protein incorporated into diet for 52 generations, and the relative resistance ratio to Cry1Ac reached 425-fold with the method of Liang et al. [18,19,22]. 2.2. Toxin source and labeling Three Cry1A proteins (Cry1Aa, Cry1Ab, and Cry1Ac) were kindly supplied by Biotechnology Research Group, Institute of Plant Protection, CAAS, and further puriWed according to the method from Garczynski et al. [23] and Luo et al. [24]. In previous studies, Cry1A (Cry1Aa, Cry1Ab, and Cry1Ac) toxicity to insect larvae was not reduced by iodination [23,25] and hence, modiWed Chloramine-T method was used to iodinate these toxins. Na125I and Chloramine-T were added to the puriWed and activated toxins separately (Na125I excess). The mixture was shaken gently for more than 1 min and the reaction was quenched with Na metabisulWte. Then the iodinated toxins were separated from free iodine using a 5-ml Sephadex G50 column. The iodinated toxins were further puriWed as follows: the radioactivity in the eluate was determined with FT-63# scintillometer and collected the hot radioactivity eluate. On the other hand, SDS–PAGE was adopted to determine the hot radioactivity of eluate. The iodinated puriWed and activated toxins were separated by SDS–PAGE, then exposed to X-ray. The exposed dot was at about 65 kDa, which suggested that these Bt toxins were well labeled (Fig. 1). The speciWc activities of labeled toxins were 2.2 mCi/mg for Cry1Aa, 2.5 mCi/ mg for Cry1Ab, and 3.1 mCi/mg for Cry1Ac (based on input toxin). 2.3. Mid-gut isolation and BBMV preparation Mid-guts of Wfth instar larvae of H. armigera were dissected longitudinally, then washed in ice-cold 0.7% NaCl buVer, and kept at ¡70 °C until used. BBMV was prepared from the mid-gut by diVerential centrifugation method of

Wolfersberger et al. [26], then frozen in liquid nitrogen, and kept at ¡70 °C until used. The concentration of proteins in BBMV preparation was determined using bovine serum albumin (BSA) as a standard by the method of Bradford [27]. 2.4. Binding of 125I-Cry1A to BBMV Binding experiments were performed as described previously [23]. For saturation binding, appropriate BBMV concentrations were selected for the subsequent competition experiments. Total binding experiments were done with either 0.5 nM (125I-Cry1Aa) or 0.1 nM (125I-Cry1Ab and 125 I-Cry1Ac) labeled toxins and various concentrations of BBMV, then they were incubated at room temperature for 1 h in 0.1 ml phosphate-buVered saline (PBS). The samples were centrifuged for 10 min at 12,000g, and the unbound toxin was removed. The pellet containing BBMV and bound toxins was washed twice with 0.5 ml PBS containing 0.1% BSA and centrifuged for 10 min at 12,000g. At the same time, nonspeciWc binding was determined by adding a 1000-fold excess of unlabeled toxin to the reaction mixtures, respectively, and the speciWc binding value was obtained through subtracting nonspeciWc binding value from the total binding value. Radioactivity was counted by a FT-63# scintillometer. Competition experiments included both homologous and heterologous competition experiments. Homologous and heterologous competition experiments were done by incubating labeled toxins and appropriate BBMV concentration, then increasing amounts of unlabeled homologous or heterologous competitor were used to compete in binding. Competition reactions were stopped by centrifugation at 12,000g for 10 min, and the radioactivity was measured as described above. 2.5. Data analysis Data from the competition experiments were analyzed using the LIGAND program [28]. Binding parameters (dissociation constant [Kd] and concentration of receptors [Rt]) were estimated from homologous competition curves as well as heterologous competition curves with the LIGAND program.

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3. Results 3.1. Binding of 125I-labeled Cry1A to BBMV The amounts of BBMV needed to reach maximum binding were diVerent for the three toxins, and they correlated directly with their in vivo activities against H. armigera. The saturation binding assays of BBMV in resistant strain were consistent with those in susceptible strain (Fig. 2A). Thus, the most toxic toxin, Cry1Ac, reached maximum speciWc binding at 50 g of BBMV per ml, while Cry1Aa and Cry1Ab maximum binding increased up to 100 g/ml (Fig. 2B). These binding data identiWed that the lowest BBMV concentration gave maximal speciWc binding. According to these data, 50 g (for Cry1Ac), 100 g (for Cry1Aa), and 100 g (for Cry1Ab) per ml were selected as the BBMV concentrations for binding competition experiments. 3.2. Competitive binding with 125I-Cry1A toxins in susceptible strain Using unlabeled Cry1Aa, Cry1Ab, and Cry1Ac as competitors, we performed homologous and heterologous binding competition experiments in susceptible strain (Fig. 3). When using 125I-Cry1Aa, not only homologous Cry1Aa but also heterologous Cry1Ab and Cry1Ac competed with high aYnity for 125I-Cry1Aa-binding sites (Table 1), which suggested that these three toxins competed for binding to one of the Cry1Aa-binding sites (Fig. 3A). Analysis of the data from homologous and heterologous binding competition experiments indicated that it Wt the one-binding-site model better than the two-binding-site model.

Fig. 3. Binding competition between 125I-Cry1Aa (A), 125I-Cry1Ab (B), and 125I-Cry1Ac (C) and unlabeled Cry1A in susceptible strain of H. armigera. (䊏) 125I-Cry1Aa, (䉱) 125I-Cry1Ab, and (䊉) 125I-Cry1Ac.

Fig. 2. Saturation binding between BBMV and Cry1A toxins. (A) Susceptible strain, (B) resistant strain; (䊏) 125I-Cry1Aa, (䉱) 125I-Cry1Ab, and (䊉) 125 I-Cry1Ac.

In the 125I-Cry1Ab competitions, only homologous Cry1Ab (Kd D 0.19 nM) and heterologous Cry1Ac (Kd D 0.84 nM) competed with high aYnity for 125ICry1Ab-binding site, while Cry1Aa (Kd D 7.8 nM) showed low aYnity (Table 1). The increasing concentrations of unlabeled Cry1Aa could not displace 125I-Cry1Ab even at high concentrations. In contrast, Cry1Ac could partly displace the labeled Cry1Ab at low concentrations (Fig. 3B). These results supported a two-binding-site model. Homologous toxin and Cry1Ac recognized this Cry1Ab-binding site. In competition experiments, only homologous Cry1Ac competed with high aYnity for 125I-Cry1Ac-binding site (Fig. 3C, Table 1). Very low competition for Cry1Aa and Cry1Ab to 125I-Cry1Ac-binding site was observed

S. Luo et al. / Pesticide Biochemistry and Physiology 85 (2006) 104–109

107

Table 1 The Kd (nM) and Rt (pmol/mg of protein) values of Cry1A toxins on BBMVs from susceptible and resistant H. armigera Toxin

Straina

125

125

I-Cry1Aa

125

I-Cry1Ab

I-Cry1Ac

Kd § SE

Rt § SE

Kd § SE

Rt § SE

Kd § SE

Rt § SE

Cry1Aa

S R

0.14 § 0.01 0.15 § 0.01

1.10 § 0.90 0.90 § 0.10

7.80 § 0.40 11.80 § 0.90

4.20 § 0. 50 12.78 § 1.20

8.40 § 0.40 11. 20 § 0.80

21.20 § 7.00 162.40 § 6.20

Cry1Ab

S R

0.86 § 0.26 1.45 § 1.10

2.40 § 0.10 5.20 § 0.80

0.19 § 0.01 0.21 § 0.03

1.38 § 0. 40 1.29 § 0.08

12.20 § 0.30 15. 40 § 2.20

32.10 § 8.20 231.20 § 2.10

Cry1Ac

S R

0.42 § 0.20 0.38 § 0.18

0.40 § 0.10 0.30 § 0.21

0.84 § 0.50 0.89 § 0.46

2.16 § 0. 30 2.28 § 0.96

0.12 § 0.01 0.20 § 0.18

5.87 § 1.40 2.23 § 0.80

a

S, susceptible strain; R, resistant strain.

(Kd D 8.40 nM for Cry1Aa and Kd D 12.20 nM for Cry1Ab), and Cry1Aa and Cry1Ab toxins did not recognize Cry1Acbinding site. 3.3. Competitive binding with 125I-labeled toxins in resistant BBMV Homologous and heterologous competitive experiments with labeled Cry1A toxins were performed with BBMV from resistant H. armigera (Fig. 4). When using 125ICry1Aa, high-aYnity competitive binding was observed with Cry1Aa and Cry1Ac (Fig. 4A). The binding aYnity of heterologous Cry1Ac (Kd D 0.38 nM) was very similar to homologous Cry1Aa (Kd D 0.15 nM). Cry1Ab (Kd D 1.45 nM) had a moderate aYnity with the Cry1Aabinding site and could only partially compete with 125ICry1Aa (Fig. 4A). Binding data analysis suggested that it Wt a one-site model better than a two or three-site model. For 125 I-Cry1Ab, Cry1Aa showed a low aYnity (Kd D 11.8 nM) that was not enough for competing with 125I-Cry1Ab, and Cry1Ac could not compete for 125I-Cry1Ab-binding sites (Fig. 4B). These binding data suggested that neither Cry1Aa nor Cry1Ac recognized Cry1Ab-binding sites, which Wt a two-site model. The Kd value of Cry1Ac homologous competition was 0.20 nM, which presented a much bigger aYnity than those for Cry1Aa and Cry1Ab (Kd D 11.20 nM for Cry1Aa and Kd D 15.40 nM for Cry1Ab) in Cry1Ac-binding site, implied a two-site model for it. 4. Discussion DiVerent resistance mechanisms to Bt in lepidopteran insects have been postulated [11,20,29,30]. In general, reduced binding of Bt toxins to the mid-gut BBMV is thought to be a primary factor [11,17,31]. In this study, the signiWcant decrease in binding of Cry1Ac in resistant strain of H. armigera is consistent with the general conclusions for other insects [31,32]. Reported data on the susceptibility of H. armigera to individual Cry protein indicate that Cry1Ac is more toxic than other Cry toxins like Cry1Ab. Estela et al. [33] suggest that the Kd value for Cry1Ac obtained from homologous competition data conWrmed its signiWcantly higher aYnity than Cry1Ab (»20-fold). However, there was no signiWcant

Fig. 4. Binding competition between 125I-Cry1Aa (A), 125I-Cry1Ab (B), and 125I-Cry1Ac (C) and unlabeled Cry1A in resistant strain of H. armigera. (䊏) 125I-Cry1Aa, (䉱) 125I-Cry1Ab, and (䊉) 125I-Cry1Ac.

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A Cry1Aa

SITE 1

B Cry1Aa

Cry1Ab

Cry1Ac

SITE 2

SITE 3

Cry1Ab

Cry1Ac

reduced (or nearly completely lost) binding of Cry1Ac with site 2, which was responsible for H. armigera resistance evolution to Cry1Ac (Fig. 5B). This phenomenon is similar to Plutella xylostella study [32]. In susceptible P. xylostella, there are two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. The investigations on resistant strains from diVerent locations show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not aVect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration aVects binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F [32]. Acknowledgments

SITE 1

SITE 2

SITE 3

Fig. 5. Binding model proposed for binding of Cry1A to sites in H. armigera mid-gut membrane from susceptible and resistant strains (A) Susceptible strain, (B) resistant strain. Dashed arrow indicates that the binding capability between site 1 and Cry1Ab decreased drastically. White part in site 2 shows that Cry1Ac-binding site has changed (B).

diVerence of Kd value between Cry1Ac and Cry1Aa/b in this study, which suggested that there was no direct correlation between toxicity and receptor binding as previous reports in other studies [21,34,35]. By contrast, the Rt values (that is, concentration of receptors) for Cry1Ac obtained from homologous competition data were signiWcantly higher than Cry1Aa and Cry1Ab in both strains. There was no signiWcant diVerence in Rt values for Cry1Aa and Cry1Ab between the resistant and susceptible strains, but for Cry1Ac in the susceptible strain it was signiWcantly higher than that in the resistant strain (>2-fold, Table 1). It was reported in lepidopterans such as Pectinophora gossypiella and Heliothis virescens that the most common type of resistance to Bt toxins (called “model 1”) entails a high level of resistance to at least one Cry1A toxin, recessive inheritance, reduced binding of at least one Cry1A toxin, and little or no cross-resistance to Cry1C [31,36]. The previous works on H. armigera indicate that there is no cross-resistance between Cry1Ac and Cry1C [37], and Cry1Ac resistance is incompletely recessive [18,38]. According to the analysis of competitive assays in the susceptible strain of H. armigera, we outlined a binding model for the pest in reference to other studies [4,32,39], which included at least three binding sites associated with binding of Cry1Aa, Cry1Ab, and Cry1Ac. In this binding model, Cry1Aa only recognized site 1, Cry1Ab did both site 1 and site 2, and Cry1Ac took all of three sites (Fig. 5A). Although there were three binding sites in the resistant strain, it was found that there was a vastly

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