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. 2021 Jan 4;87(2):e01930-20. doi: 10.1128/AEM.01930-20

Bacillus thuringiensis Cry1Ab Domain III β-16 Is Involved in Binding to Prohibitin, Which Correlates with Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae)

Igor Henrique Sena da Silva a, Isabel Gómez b, Sabino Pacheco b, Jorge Sánchez b, Jie Zhang c, Tereza Cristina Luque Castellane d, Janete Aparecida Desiderio d, Mario Soberón b, Alejandra Bravo b,✉, Ricardo Antônio Polanczyk a
Editor: Harold L Drakee
PMCID: PMC7783339  PMID: 33127814

Helicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis. In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify β-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

KEYWORDS: Cotton bollworm, Helicoverpa armigera, Bacillus thuringiensis, Cry toxins, mechanism of action, cadherin, PHB, prohibitin

ABSTRACT

Helicoverpa armigera is a major insect pest of several crops worldwide. This insect is susceptible to some Bacillus thuringiensis (Bt) Cry insecticidal proteins expressed in transgenic crops or used in biopesticides. Previously, we identified H. armigera prohibitin (HaPHB) as a Cry1Ac-binding protein. Here, we further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), well recognized as a Cry1Ac receptor. HaPHB-2 midgut protein and HaCAD toxin-binding region (TBR) fragment from H. armigera were expressed in Escherichia coli cells, and binding assays with different Cry1 toxins were performed. We demonstrated that Cry1Ab, Cry1Ac, and Cry1Fa toxins bound to HaPHB-2 in a manner similar to that seen with HaCAD-TBR. Different Cry1Ab mutant toxins located in domain II (Cry1AbF371A and Cry1AbG439D) or domain III (Cry1AbL511A and Cry1AbN514A), which were previously characterized and found to be affected in receptor binding, were analyzed regarding their binding interaction with HaPHB-2 and toxicity against H. armigera. One β-16 mutant (Cry1AbN514A) showed increased binding to HaPHB-2 that correlated with 6-fold-higher toxicity against H. armigera, whereas the other β-16 mutant (Cry1AbL511A) was affected in binding to HaPHB-2 and lost toxicity against H. armigera. Our data indicate that β-16 from domain III of Cry1Ab is involved in interactions with HaPHB-2 and in toxicity. This report identifies a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect, suggesting that this protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigera.

IMPORTANCE Helicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis. In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify β-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

INTRODUCTION

The cotton bollworm, Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), is a polyphagous insect pest worldwide. In Brazil, the first report of this insect was registered in 2013/2014, attacking soybean crops in Goiás and Bahia states and cotton in Mato Grosso state (1–3). Its control costs more than 2 billion U.S. dollars annually worldwide (4). The most important challenge is related to its capacity to evolve resistance to chemical insecticides (5–6) and to biopesticides such as the insecticidal proteins produced by Bacillus thuringiensis (Bt) bacterium (7–9).

Bt is a Gram-positive bacterium that produces different insecticidal toxins that show toxicity against several insect species that are of agricultural importance or are vectors of human diseases (10). These proteins have been used worldwide, showing several advantages compared to chemical control strategies since they are highly specific against target pests, are harmless to vertebrates, and are biodegradable (11–13).

The mode of action of Cry proteins produced by Bt is a complex process involving the interaction of these toxins with different receptors in the larval midgut epithelium, triggering toxin oligomerization and insertion of the oligomer into the membrane and resulting in pore formation in the apical membrane of the midgut cells and insect death (14–15). Several studies have identified in different lepidopteran insects the presence of potential Cry toxin receptors, including cadherin-like (CAD) proteins; glycosylphosphatidylinositol (GPI)-anchored aminopeptidases (APN); alkaline phosphatases (ALP); polycalin, a 270-kDa glycoconjugate; a 250-kDa protein named P252; an α-amylase; and members of the ATP-binding cassette (ABC) transporter protein family, such as ABCC2, ABCC3, and ABCA2 transporters (9, 16–23). However, it is still possible that additional proteins could be involved in the mode of action of Cry toxins.

The most common mechanisms of resistance to Cry toxins are mutations linked to Cry toxin receptors that result in reduced toxin binding (24, 25).

Recently, we analyzed the Cry1Ac-binding proteins in the early and late instars of H. armigera by means of pulldown assay followed by protein identification by liquid chromatography-mass spectrometry (LC-MS) analysis (26). Among the proteins identified, H. armigera prohibitin-2 (HaPHB-2) was identified as a Cry1Ac-binding protein preferentially expressed in the early instars (26). PHB is composed of prohibitin-1 (PHB-1) and prohibitin-2 (PHB-2) proteins that share more than 50% identity and are able to form heteroligomers (27). A PHB-1 that binds Cry4Ba was previously identified in Aedes aegypti (28), and also it was shown that Cry3Aa was able to bind both PHB-1 and PHB-2 in larvae of the coleopteran Leptinotarsa decemlineata (29). In addition, PHB proteins may function as receptors for Vi capsular polysaccharide from Salmonella enterica serovar Typhi in intestinal epithelial human cells and for dengue virus in A. aegypti cells (30–31).

In this work, we analyzed the binding interaction of HaPHB-2 with different Cry1 toxins and their toxicity against H. armigera. The HaPHB-2 midgut protein was cloned and expressed in Escherichia coli cells to perform semiquantitative binding assays in comparison to a previously described HaCAD toxin-binding region (TBR) fragment since HaCAD was previously shown to be a functional receptor of Cry1Ac in H. armigera (32, 33). Furthermore, we found that β-16 from domain III of Cry1Ab participates in the binding interaction of Cry1Ab toxin with HaPHB-2 and is involved in Cry1Ab toxicity against H. armigera. This report characterizes the HaPHB-Cry1A interaction, supporting the idea that HaPHB-2 also participates in the mechanism of action of Cry1Ab toxin in H. armigera.

RESULTS

For cloning of HaPHB-2, we took advantage of the previously annotated PHB-2 protein sequence from H. armigera (GenBank accession number XM_021345859). HaPHB-2 is a 299-amino-acid protein with a predicted molecular weight of 33 kDa.

HaPHB protein competes with Cry1Ac in binding to H. armigera BBMV.

In order to analyze the binding of Cry1Ac to H. armigera brush border membrane vesicle (HaBBMV), qualitative binding assays were performed. Cry1Ac binds HaBBMV in a toxin concentration-dependent manner (Fig. 1, left panel). The analysis of the densitometry of the bands shown in Fig. 1A performed by the use of ImageJ software revealed an apparent binding affinity (Kd) of 8.51 ± 1 nM (Fig. 1, right panel).

FIG 1.

FIG 1

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Binding interaction of Cry1Ac toxin binding with BBMV from H. armigera. (Left panel) A total of 10 μg of HaBBMV from the 3rd larval instar was incubated with 2.5 to 20 nM Cry1Ac toxin (lanes 2 to 5). The HaBBMV and bound toxin were separated by centrifugation, suspended in running buffer, and separated by SDS-PAGE electrophoresis. Proteins were blotted into a PVDF membrane, and the Cry1Ac toxin bound to the HaBBMV was recognized with anti-Cry1Ac antibody and revealed with a secondary anti-rabbit antibody coupled to HRP as described in Materials and Methods. A negative control consisting of HaBBMV without toxin incubation (lane 1) and a positive control with 2.5 nM Cry1Ac toxin loaded directly into the SDS-PAGE (lane 6) were included in the experiment and are shown in the figure. The optical density of the 65-kDa band was measured by using ImageJ software. Size markers (SM) were Precision Plus all blue prestained protein standards (Bio-Rad). (Right panel) Analysis of the densitometry of the bands accordingly to the concentration of protein used in the binding assay.

To determine if HaPHB-2 is involved in the binding of Cry1Ac to HaBBMV, the recombinant HaPHB-2 protein was purified from E. coli cells and used as the competitor in Cry1Ac binding competition assay of binding to HaBBMV (Fig. 2). A 5 nM concentration of Cry1Ac toxin was incubated with 10 μg of HaBBMV in the absence or in the presence of different fold molar excesses of HaPHB-2 (100 to 500). A reduction of toxin binding to HaBBMV was observed in the presence of HaPHB-2 in a concentration-dependent way (Fig. 2). Densitometry analysis of the bands performed by the use of ImageJ software showed 2.8-fold and 13.2-fold reductions of Cry1Ac binding in the presence of 100-fold and 200-fold molar excesses of HaPHB-2, respectively, while a complete competition of binding was observed with 500-fold molar units of HaPHB-2 used as the competitor.

FIG 2.

FIG 2

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Competition binding assays of binding of Cry1Ac to HaBBMV using HaPHB as the competitor. Binding of 5 nM Cry1Ac toxin to 10 μg HaBBMV protein from 3rd instar larvae was done in the absence and presence of 100-fold to 500-fold molar excesses of HaPHB-2 (lanes 2 to 5). The HaBBMVs with the bound toxin were separated by centrifugation, suspended in running buffer, and separated in SDS-PAGE. Proteins were blotted to a PVDF membrane, and the toxin bound to the HaBBMV was recognized with anti-Cry1Ac antibody and revealed with secondary anti-rabbit antibody coupled to HRP as described in Materials and Methods. Two controls were included, a negative one using HaBBMV without incubation with toxin (lane 6) and a positive one using 2.5 nM Cry1Ac toxin directly loaded in the SDS-PAGE (lane 7). The optical density of the 65-kDa band was measured by using ImageJ software. Size markers (SM) were Precision Plus all blue prestained protein standards (Bio-Rad).

Cry1Ab, Cry1Ac, and Cry1Fa bind similarly to HaPHB-2 and HaCAD proteins from H. armigera.

To further analyze the interaction of different Cry1 proteins with HaPHB-2, we determined the levels of binding of Cry1Ab, Cry1Ac, and Cry1Fa toxins to heterologously expressed HaPHB-2 proteins by enzyme-linked immunosorbent assays (ELISAs) of binding. As a control, we included a HaCAD fragment that was reported to contain the toxin-binding region (TBR) (33). Levels of binding of Cry1 toxins to both proteins, HaCAD-TBR and HaPHB-2, showed that they were saturable, and Cry1Ac and Cry1Fa showed slightly higher binding to both receptors than Cry1Ab did (Fig. 3). The analysis of the apparent binding affinities obtained after total binding analysis showed that Cry1Ac and Cry1Fa toxins interacted with HaCAD-TBR with a 2-fold-higher apparent level of binding (Cry1Ac Kd = 4.65 ± 1.6 nM and Cry1Fa Kd = 5.46 ± 0.9 nM) than Cry1Ab (Kd = 9.12 ± 0.72 nM) (P value of <0.04, which is considered to be statistically significant) (Fig. 3A). However, specific binding analysis is still required to be performed in order to confirm these data. A similar pattern was observed in the interaction of Cry toxins with HaPHB-2, since Cry1Ac and Cry1Fa presented slightly higher apparent levels of binding (Cry1Ac Kd = 6.65 ± 1.84 nM and Cry1Fa Kd = 7.75 ± 1.86 nM) than Cry1Ab (Kd = 9.68 ± 0.89 nM) However, the P values of <0.39 determined from these data indicated that these differences were not statistically significant (Fig. 3B).

FIG 3.

FIG 3

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Binding ELISAs of Cry1A toxins to the recombinant HaCAD and HaPHB-2 proteins from H. armigera expressed in E. coli cells. The recombinant proteins were immobilized on ELISA plates, incubated with Cry1Fa, Cry1Ab, and Cry1Ac toxins (0 to 200 nM), and detected with anti-Cry1Ab, anti-Cry1Ac, or anti-Cry1Fa and HRP-conjugated anti-rabbit antibody as secondary antibody. The absorbance was determined at 490 nm, and the data were analyzed by using GraphPad Prism 7 software to calculate the dissociation constant (Kd) by Scatchard analysis. Different asterisks indicate statistically significantly different data determined by one-way ANOVA (P value < 0.05).

In addition, ligand blot analyses of binding of Cry1Ac toxin to the recombinant HaPHB-2 and HaCAD-TBR proteins from H. armigera purified from E. coli cells were performed (Fig. 4). We analyzed the binding of 10 nM Cry1Ac to different amounts of the purified HaCAD-TBR protein of 47.5 kDa (Fig. 4A) and to the purified HaPHB-2 protein fused to SUMO protein, resulting in a 45-kDa protein (Fig. 4B). The images in Fig. 4 show that Cry1Ac bound to these receptor proteins.

FIG 4.

FIG 4

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Ligand blot assays showing binding of Cry1Ac toxin to HaCAD-TBR and HaPHB-2 recombinant proteins from H. armigera. Cry1Ac (10 nM) was bound to different amounts of HaCAD-TBR fragment (A) or HaPHB-2 fragment (B). Bound Cry1Ac protein was revealed by using anti-Cry1Ac antibody and HRP-coupled anti-rabbit secondary antibody. Size markers (SM) were Precision Plus all blue prestained protein standards (Bio-Rad).

Correlation of binding of β16 mutants to HaPHB-2 protein and their toxicity against H. armigera.

To identify the toxin regions involved in the interaction with HaPHB-2 and the role of HaPHB-2 in toxicity, we characterized the binding to HaPHB-2 and toxicity against H. armigera larvae of different domain II and domain III Cry1Ab mutants previously characterized as affected in binding to different receptor molecules in Manduca sexta or Spodoptera frugiperda. Regarding Cry1Ab domain II mutations, we chose to work with two mutants affected in toxicity against M. sexta. The first was a Cry1AbF371A mutant, located in loop 2, whose oligomeric structure was previously shown to be affected in binding to M. sexta APN and ALP receptors but whose monomeric structure was not found to be affected in binding to APN or ALP (34). Also, we analyzed binding of Cry1AbG439D, located in loop 3, which was affected with respect to binding to a M. sexta CAD repeat 12 fragment (CR12) and also to M. sexta ALP and APN1 (35). In the case of domain III mutations, two Cry1Ab β-16 mutants were analyzed. Mutant Cry1AbL511A was previously shown to be severely affected in binding interactions with M. sexta ALP and APN receptors and also affected in toxicity to M. sexta larvae (34), while the Cry1AbN514A mutant was shown to have significantly higher toxicity against S. frugiperda larvae but a similar level of toxicity against M. sexta and its increased toxicity was shown to correlate with increased binding to S. frugiperda CAD, ALP, and APN1 proteins (36).

Comparative analyses of binding of these four Cry1Ab mutants to HaCAD-TBR and HaPHB-2 were performed using nonsaturation conditions in ELISAs, where the binding of a 5 nM concentration of each toxin mutant to these two receptors was analyzed. The data in Fig. 5 show that none of these Cry1Ab mutants was affected in binding to a HaCAD-TBR fragment. In contrast, we found that a Cry1AbL511A mutant showed a reduction in binding to HaPHB-2 and that the rest of the mutants showed similar levels of binding to this protein under these binding conditions (Fig. 5). Specific binding remains to be analyzed.

FIG 5.

FIG 5

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Comparative binding analyses of Cry1Ab mutants to HaCAD-TBR and HaPHB-2. Binding ELISAs were performed using nonsaturation conditions. Purified recombinant HaPHB-2 (right panel) or HaCAD-TBR (left panel) protein fragments (1 μg/well) were used to coat 96-well ELISA plates. The binding of a 5 nM concentration of each toxin mutant to these two receptors was analyzed and detected with anti-Cry1Ab antibody as primary antibody and HRP-conjugated anti-rabbit antibody as secondary antibody. The absorbance was determined at 490 nm, and the data were analyzed by using GraphPad Prism 7 software. Different letters indicate statistically significant differences determined by one-way ANOVA (P value < 0.05).

To further confirm these data, additional ELISAs of binding of these four Cry1Ab mutants to HaPBH-2 were performed. The data revealed that Cry1AbN514A gained higher apparent binding to HaPHB-2 (Kd = 2.54 ± 1.16 nM) than the Cry1Ab toxin, which showed a Kd value of 7.12 ± 0.89; while Cry1AbL511A showed less binding to HaPHB-2 (Kd = 13.12 ± 0.72 nM), and analysis of variance (ANOVA) of these data indicated that these differences were statistically significantly different (P < 0.05) (Fig. 6). Note that total binding of the Cry1AbL511A mutant to HaPHB-2 was reduced significantly. Interestingly, the binding of Cry1AbN514A or Cry1AbL511A mutants correlated with their toxicity against neonate H. armigera larvae, since the Cry1AbN514A mutant was ∼6-fold more toxic (50% lethal concentration [LC50] = 43 ng/cm2 [fiducial limit, 30 to 62]) than Cry1Ab, showing a 50% lethal concentration (LC50) value of 260 ng/cm2 (fiducial limit, 178 to 398), while the Cry1AbL511A mutant lost toxicity, showing at least 20-fold-lower toxicity (LC50 value of >5,000 ng/cm2) than Cry1Ab (Table 1). The two domain II mutants, Cry1AbF371A and Cry1AbG439D, bound to HaPHB-2 with similar apparent binding levels (Kd= 6.65 ± 0.61 nM and Kd = 6.98 ± 0.78 nM, respectively; P values of <0.05) (Fig. 6). Table 1 shows that the Cry1AbG439D mutant was affected in insecticidal activity, while Cry1AbF371A was not.

FIG 6.

FIG 6

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ELISA of binding of different Cry1Ab mutants to recombinant HaPHB-2 protein expressed in E. coli cells. The recombinant HaPHB-2 protein was immobilized on ELISA plates, incubated with different variants of Cry1Ab as indicated in the figure (0 to 200 nM), and detected with anti-Cry1Ab antibody and HRP-conjugated anti-rabbit antibody. The absorbance was determined at 490 nm, and the data were analyzed by using GraphPad Prism 7 software to obtain relative binding affinities (Kd) by Scatchard analysis. Different asterisks indicate statistically significantly different data analyzed by one-way ANOVA (P value < 0.05). Wt, wild type.

TABLE 1.

Insecticidal activity of Cry1Ab wild-type and domain II and III mutants against H. armigera larvae

Toxin LC50, ng/cm2
(fiducial limit)a 		Slopeb χ²c
Cry1Ab 260 (178–398) 1.16 ± 0.19 1.63
Cry1AbL511A >5,000 1.25 ± 0.20 0.93
Cry1AbN514A 43 (30–62) 1.30 ± 0.20 1.04
Cry1AbG439D >2,000 1.06 ± 0.21 1.24
Cry1AbF371A 244 (97–445) 1.36 ± 0.21 6.36
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a

Data represent the concentration killing 50% of the population, with 95% fiducial limits indicated in parentheses. Units are nanograms of Cry toxin per square centimeter of artificial diet (25 to 5,000 ng/cm2).

b

Data represent slope ± standard error.

c

The chi-square values (P < 0.05) did not reach statistical significance, indicating that the data fit the Probit analysis model.

DISCUSSION

It has been demonstrated that H. armigera shows differing levels of susceptibility to different Cry toxins. Among 24 Cry toxins analyzed, 7 (Cry1Ac, Cry1Ah, Cry1Fa, Cry2Aa, Cry2Ab, Cry2Ac, and Cry2Af) were previously shown to be active against H. armigera, while 4 other Cry toxins (Cry1Aa, Cry1Ab, Cry1Ia, and Cry9Aa) were classified as potentially having toxicity (37). Identification of the midgut binding proteins that participate in Cry toxicity is crucial to further understand their function in the mechanism of action of these Cry proteins. Here, we evaluated the binding capacity of three Cry1 toxins to purified HaPHB-2 and HaCAD-TBR proteins from H. armigera that were previously identified as Cry1Ac-binding proteins (7, 26, 32, 33, 38, 39).

We demonstrated that Cry1Ac binds to H. armigera BBMV with high affinity, which confirms results of previous analyses of binding of Cry1Ac to BBMV from this insect pest (40–41). The Cry1Ac protein is one of the most active toxins against H. armigera and has been widely used in transgenic Bt cotton (4, 42). We previously identified HaPHB-2 as a binding protein for this toxin by pulldown assay and liquid chromatography-mass spectrometry sequencing in 2nd instar H. armigera midgut larvae (26). Here, we show that a 500-fold-higher molar concentration of HaPHB-2 protein competed with the binding of Cry1Ac to HaBBMV, supporting the idea that HaPHB-2 is able to titrate Cry1Ac binding at a large excess, indicating that it may participate as a receptor protein of Cry1Ac toxin in H. armigera, although more studies are needed to uncover its specific role in the mechanism of action of Cry proteins. It is possible that HaPHB-2 could also compete with the binding of the toxin to other receptors, since it has been shown that the Cry1Ab domain III β-16 region is also involved in binding to M. sexta APN and ALP receptors (34). Thus, it is possible that stearic hindrances may affect binding to those other receptors, resulting in a significant reduction in Cry1Ac binding in the presence of that high excess of HaPHB-2 protein. Also, it is known that CAD receptor is much less abundant than other receptors such as APN (43); thus, this competition could also reflect the differential expression levels of Cry receptors in HaBBMV.

Our work revealed that PHB-2 functions as a Cry1-binding protein in lepidopteran insects. Previously, PHB was identified as a Cry binding protein in two other insect orders, i.e., A. aegypti (Diptera) for Cry4Ba (28) and L. decemlineata (Coleoptera) for Cry3Aa (29). Studies of silencing of PHB-1 by RNA interference (RNAi) in L. decemlineata showed that PHB-1 is an essential protein, since its silencing affected the viability of the larvae (29).

Using binding ELISAs, we demonstrated that Cry1Ac, Cry1Fa, and Cry1Ab toxins bound to HaCAD and HaPHB-2 with similar apparent levels of binding at the nanomolar range and that the binding of Cry1Ac to HaCAD-TBR and HaPHB-2 protein was further confirmed by ligand blotting assays. Disruption of toxin binding to larval midgut receptors is the most common mechanism of resistance of target insects to Cry toxins (24). Mutations in CAD were reported to be involved in Cry1Ac resistance in several lepidopteran larvae, such as those of Heliothis virescens (44), Helicoverpa zea (45), Pectinophora gossypiella (46–48), T. ni (49), Chilo suppressalis (50), and H. armigera (32, 33, 38).

To analyze the toxin regions possibly involved in binding of Cry1 toxins to HaPHB-2, we made use of a series of Cry1Ab domain II or domain III mutants that were previously shown to affect binding to different receptors as CAD, ALP, and APN in M. sexta or S. frugiperda (34–36, 51). Domain II loop 2 (Cry1AbF371A) or loop 3 (Cry1AbG439A) mutants were able to bind to HaPHB-2, while domain III β-16 mutants (Cry1Ab-N514A and Cry1Ab-L511A) showed a correlative effect on binding to HaPHB-2 and toxicity to H. armigera. Mutant Cry1AbN514A showed 6-fold-higher toxicity than Cry1Ab against H. armigera, which correlated with its apparent binding to HaPHB-2 being higher than that seen with Cry1Ab, while Cry1AbL511A was found to be severely affected in toxicity, which directly correlated with a lower level of binding interaction with HaPHB-2 (Fig. 6; see also Table 1). These results indicate that the domain III β-16 region of Cry1Ab toxin plays an important role in binding interactions with PHB-2 and suggest that H. armigera HaPHB-2 could be a functional receptor of Cry1Ab and Cry1Ac toxins. However, we cannot rule out the possibility that other regions of the toxin, such as domain II exposed loops, are also involved in binding to HaPHB-2, since only two domain II loop mutants were analyzed. Also, we cannot rule out the possibility that Cry1Ab domain III is involved in binding to other midgut receptors, which would also explain its defects in toxicity. This remains to be analyzed.

PHB-2 is a conserved protein in divergent species from prokaryotes and eukaryotes, and this protein is typically associated with lipid rafts in eukaryotic cells (52). Lipid rafts are membrane microdomains rich in cholesterol and sphingolipids. GPI-anchored proteins are selectively located in lipid rafts and are implicated in different processes in the membrane, such as ion channel regulation, membrane protein chaperoning, vesicle and protein trafficking, membrane-cytoskeleton coupling, formation of specialized membrane structures, and several cell-signaling responses (53–55). APN and ALP are both GPI-anchored proteins, and it was previously shown that these proteins are selectively included in lipid rafts from M. sexta and in H. virescens midgut cells (56–58). The APN and ALP proteins have been identified as Cry toxin receptors that are present in several lepidopteran insects (17, 59, 60). Since the domain III β16 region of Cry1Ab has been shown to be involved in the binding interaction of this protein with ALP or APN, facilitating Cry1 toxin oligomer membrane insertion (34, 51), we speculate that PHB-2 may have a similar role in the Cry toxin mode of action. However, this hypothesis remains to be evaluated in the future.

Regarding the domain II mutants analyzed, the toxicity data showed that the Cry1AbG439D mutant, located in loop 3 of domain II, lost toxicity against H. armigera but was not affected in binding to HaPHB-2. It was previously reported that the loop 3 region of Cry1Ab and Cry1Ac is an important region involved in binding interaction with multiple receptors from M. sexta since it binds to the CAD repeat 12 fragment (CR12) and also to M. sexta ALP and APN proteins (35, 61). Thus, it is possible that this mutant is affected in toxicity against H. armigera due to its defects in binding interactions with those other receptors. Our binding ELISAs showed that Cry1AbG439D was not affected in HaCAD-TBR binding (Fig. 5). It was reported before that Cry1AbG439D mutant was specifically affected in its binding interaction with one region of CAD protein in M. sexta that corresponds to the CR12 region (35). Nevertheless, the HaCAD-TBR analyzed here contains two Cry1Ab binding sites and thus the other Cry1Ab binding site may have compensated for binding to HaCAD-TBR in these in vitro assays, explaining the observed interaction. It is also possible that the defect in the toxicity of this mutant against H. armigera could be due to lower binding to other receptors, such as HaABCC2, which has been shown to be important for Cry1Ac toxicity in H. armigera (8).

This report shows characterization of the binding interaction of Cry1 toxins with HaPHB-2 from H. armigera, supporting the idea that it represents a novel binding site in lepidopteran pests, although its specific role in the mechanism of action of Cry toxins and the potential mechanism of resistance of these toxins still remain to be elucidated.

MATERIALS AND METHODS

Expression, purification, and activation of Cry1A toxins.

Bt serotype kurstaki strain HD-73 cells expressing Cry1Ac or crystalliferous Bt strain 407 cells expressing Cry1Ab (62) or Cry1Fa (63) proteins or Cry1Ab mutant proteins (G439D, F371A, N514A, and L511A) (34–36, 51) were grown at 30°C to complete sporulation for 3 days in nutrient broth sporulation medium (64). For Bt strain 407 expressing Cry1Ab and Cry1Fa proteins, the growth medium was supplemented with erythromycin at 10 μg ml−1. Spores/crystals were washed three times in 0.3 M NaCl–10 mM EDTA, pH 8.0. Crystal inclusions were solubilized in an alkaline buffer (50 mM Na2CO3, NaHCO3, 0.2% β-mercaptoethanol, pH 10.5) for 1 h at 37°C. Trypsin-activated toxins were obtained by treatment of soluble protoxins with trypsin (N-tosyl-l-phenylalanine chloromethyl ketone [TPCK]-treated trypsin from bovine pancreas; Sigma Aldrich, St. Louis, MO, USA) in a mass ratio of 1: 20 (trypsin/toxin) for 1 h at 37°C. Phenylmethylsulfonyl fluoride (PMSF) (1 mM final concentration) was added to stop proteolysis. Finally, the Cry toxins were purified with a high-performance (HP) HiTrap ion-exchange chromatographic column (GE Healthcare) according to the manufacturer's protocol. The final concentrations of the purified proteins were determined by the Bradford method using bovine serum albumin (BSA) as a standard. Figure S1 shows the different Cry proteins used in this study after SDS-PAGE electrophoresis.

Midgut dissection and BBMV purification from H. armigera larvae.

The H. armigera larvae used were from a laboratory colony maintained at the School of Agricultural and Veterinarian Sciences, São Paulo State University, Jaboticabal, SP, Brazil. Insects were reared on an artificial diet (65). H. armigera midgut tissues from 3rd instar larvae were dissected and stored immediately at −70°C. Brush border membrane vesicles (BBMV) were prepared by the magnesium precipitation method without addition of protease inhibitors as described previously by Wolfersberger (66) and stored at −70°C until use. The BBMV protein concentration was determined with the Lowry DC protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (Pierce) as a standard. The enrichment of APN in BBMV was determined as previously reported (26), showing that APN activity was 13-fold higher in BBMV than the initial homogenate.

Heterologous expression of HaPHB-2 and HaCAD proteins from H. armigera in E. coli cells.

The HaCAD gene used in this work (GenBank accession number JN836550) was previously characterized, and a 1,097-bp gene fragment containing the HaCAD toxin-binding region was previously cloned in pET22b (33, 67). A HaPHB-2 gene (GenBank accession number XM_021345859) from H. armigera larvae was cloned in pET SUMO (Thermo Scientific, Waltham, MA) and heterologously expressed in E. coli cells. For cloning gene fragments, the total RNA from 3rd instar H. armigera midgut tissue was extracted using an RNeasy minikit (Qiagen). For HaPHB-2 amplification, specific primers (PHB-For [5′ ATG GCA CAA AGT AAG CTT 3′] and PHB-Rev [5′ TTA CTT AGT CAG TTT C 3′]) were designed. The PCR product containing the full-length HaPHB-2 (900-bp) gene (GenBank accession number XM_021345859) from H. armigera larvae was ligated into pET SUMO vector (Thermo Scientific, Waltham, MA). The PCR products were adenylated with a final extension at 72°C for 30 min to ensure that all PCR products were of full length and were 3′ adenylated. A ligation reaction was performed overnight at 15°C according to the instructions of the manufacturer. The construction was transformed into E. coli BL21(DE3) cells (Invitrogen, Carlsbad, CA). The presence of pET SUMO vector should increase the protein yield, and the SUMO gene that was fused to a HaPHB-2 gene increased the solubility of the fusion protein, resulting in a protein that was 45 kDa in size. Plasmid was purified and DNA sequence was confirmed at the facilities of the Institute of Biotechnology, Universidad Nacional Autónoma de Mexico (UNAM). E. coli HaCAD and HaPHB transformant strains were grown overnight with agitation at 37°C in 5 ml of LB broth containing 100 μg ml−1 of ampicillin for HaCAD and kanamycin for HaPHB-2. A total of 100 μl from overnight cultures was inoculated into 100 ml of 2× TY broth (tryptone 16 g liter−1, yeast extract 10 g liter−1, NaCl 5 g liter−1, 0.1% glucose) supplemented with the appropriate antibiotic, and the reaction mixture was placed in a 250-ml flask and incubated until an optical density at 600 nm (OD600) of 0.7 was reached. Protein synthesis was induced by addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the reaction mixture was further incubated for 6 h at 30°C. The culture was centrifuged for 15 min at 2,795 × g at 4°C, and the pellet was suspended in STE buffer (10 mM Tris-HCl, 1 mM EDTA, 8 M urea, pH 8). After sonication (5 min on ice, with 1-min pulses) and centrifugation (12,850 × g for 5 min at 15°C), the supernatant was recovered and subjected to affinity purification using nickel-agarose beads (Qiagen). The column was washed first with 30 ml of 1× phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) to dilute the urea and then with 5 ml of 35 mM imidazole in PBS buffer (pH 7.4) and was finally eluted with 3 ml 250 mM imidazole. The final sample was dialyzed against PBS buffer.

Binding of Cry1Ac toxin to H. armigera BBMV.

Binding of Cry1Ac activated toxin to HaBBMV was performed by incubating 2.5 to 20 nM purified toxin with 10 μg HaBBMV protein for 1 h at room temperature in 100 μl of binding buffer (PBS, 0.1% BSA, 0.1% Tween 20, pH 7.6). A control consisting of HaBBMV without toxin was included in the assay. After incubation, the unbound toxin was removed by centrifugation for 10 min at 12,850 × g. The pellet containing HaBBMV and bound toxin was washed twice with 100 μl binding buffer, suspended in 10 μl of PBS, and mixed with 10 μl of sample-loading 2× Laemmli buffer (0.125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.01% bromophenol blue). Samples were boiled 3 min, loaded in 10% SDS-PAGE gels, and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Bio-Vin). The PVDF membrane was blocked with BSA 0.5% for 1 h under conditions of agitation, and bound Cry1Ac toxin was revealed by Western blotting using anti-Cry1Ac antibody (1/10,000 dilution; 1 h) as the primary antibody. As the secondary antibody, a goat anti-rabbit antibody coupled to horseradish peroxidase (HRP) was used (Santa Cruz Biotechnology, Dallas, TX, USA) (1/10,000 dilution; 1 h), followed by Luminol treatment (Santa Cruz Biotechnology Inc.), according to the manufacturer’s instructions.

Competition assays of Cry1Ac toxin binding to BBMV with HaPHB-2.

Heterologous competition binding assays of Cry1Ac toxin binding to HaBBMV were done using different concentrations of HaPHB-2 as the competitor. For these assays, 10 μg of BBMV was incubated with 5 nM activated Cry1Ac toxin in the presence of different molar excesses (100-fold, 200-fold, and 500-fold) of HaPHB-2 in 100 μl of binding buffer at room temperature for 1 h. Unbound proteins were removed by centrifugation at 12,850 × g for 10 min at 4°C and were washed twice. The HaBBMV pellet containing the bound proteins was suspended in 10 μl H2O, mixed with 10 μl of 2× Laemmli sample buffer, and boiled for 3 min. Samples were loaded on a 12% SDS-PAGE gel and electrotransferred to a PVDF membrane. The membrane was blocked with 1× PBS–Tween 20 (2%) and maintained under conditions of agitation for 1 h, and bound protein was recognized using anti-Cry1Ac antibody (1:20,000 dilution) and a secondary goat anti-rabbit antibody coupled to HRP (1:20,000 dilution), followed by Luminol treatment as described above.

Binding ELISAs.

Purified recombinant HaPHB-2 or HaCAD protein fragments were used to coat 96-well ELISA plates (Rochester, NY, USA) (1 μg/well). Activated Cry1Ab, Cry1Ac, and Cry1Fa mutants or domain II and III Cry1Ab mutants at different concentrations (0 to 200 nM) were incubated with the receptor-coated ELISA plates. Comparative analyses were also performed with the different Cry1Ab mutants used under nonsaturated conditions, and each mutant protein (5 nM) was compared with 5 nM Cry1Ab toxin. Unbound toxin was removed with PBS followed by three washes with PBS supplemented with 0.1% Tween 20. Bound toxins were detected using the corresponding polyclonal antibodies (anti-Cry1Ac, anti-Cry1Ab, and anti-Cry1Fa) (1:20,000 dilution) and secondary goat anti-rabbit antibody conjugated with HRP enzyme (1:20,000 dilution). Finally, o-phenylenediamine (Sigma) and H2O2 were used as substrates for peroxidase activity detection. Reactions were stopped by adding 50 μl of 5 M HCl, and OD490 was measured using an ELISA microplate reader (PerkinElmer, Waltham, MA, USA). Negative controls, where the HaPHB-2 or HaCAD proteins were not used to coat the ELISA plate wells, were performed in parallel. These negative controls gave values that were lower than 7% of the values seen in experimental assays performed with Cry1Ab and the mutants Cry1AbN514A, Cry1AbG439D, and Cry1AbF371A. Only mutant Cry1AbN511A showed a negative-control value of 12%. The data shown here were obtained after subtracting the data from the negative controls from the data from the samples containing the receptors. All experiments were done in triplicate. Comparisons of binding data were analyzed by t test using GraphPad Prism 7 (version 5.0b), and analyses of Scatchard plots were used to obtain relative apparent binding affinity (Kd) values. Data of the binding ELISAs were analyzed by using one-way analysis of variance (ANOVA), showing significant differences (P < 0.05).

Ligand blot assays.

Different amounts of HaPHB-2 and HaCAD proteins were separated by SDS-PAGE (12% acrylamide) and transferred to a PVDF membrane. After renaturation and blocking, the blots were incubated for 1 h with 10 nM Cry1Ac toxin in washing buffer (0.5% Tween 20–1× PBS) at room temperature. Unbound toxin was removed by three washes in washing buffer performed for 10 min per wash, and bound toxin was identified by Western blotting after incubating the blots with anti-Cry1Ac antibody (1:10,000 dilution; 1 h). As secondary antibody, a goat anti-rabbit secondary antibody coupled to HRP (1:10,000 dilution; 1 h) was used, followed by Luminol treatment as described above.

Toxicity bioassays.

Toxicity bioassays were performed with H. armigera neonate larvae by the surface contamination method. Different doses of crystals/spores were applied to the diet surface contained in 128-well polystyrene plates (Bio-BA-128 bioassay trays; C-D International, Inc.). Six different doses of crystals/spores (from 25 to 5,000 ng/cm2) were assayed. A total of 48 larvae per toxin concentration (1 larva per well) were used. The negative control was larvae that had a diet without added toxin. The plates were incubated at 26°C with 65% ± 5% relative humidity and a 14-h light/10-h dark cycle. These toxicity bioassays were performed in triplicate. Mortality results were recorded after 7 days; larvae were considered dead if no movement was apparent after the larvae were touched. The 50% lethal concentration (LC50) was estimated by Probit analysis (Polo-PC LeOra Software). The fiducial limits were estimated for each LC50 value, and the results are shown in Table 1.

Statistical analysis.

Kd values and standard errors were determined from Scatchard plot analysis of ELISAs, and P values representing statistical significance were determined for each analysis. Statistically significant differences between results of the binding ELISAs (Fig. 3 and 6) were determined by using one-way analysis of variance (ANOVA). P values of <0.05 represent statistical significance. Data were analyzed using GraphPad Prism 7 (version 5.0b).

Supplementary Material

Supplemental file 1
AEM.01930-20-s0001.pdf (465.3KB, pdf)

ACKNOWLEDGMENTS

We thank the São Paulo Research Foundation (FAPESP; grants 2018/13974-7, 2019/00264-4), Dirección general de asuntos del personal académico/Universidad Nacional Autónoma de Mexico (DGAPA/UNAM IN203619) and Consejo Nacional de Ciencia y Tecnología (CONACyT A1-S-12053) for financial support of the work reported here.

I.G., A.B., and M.S. are authors of a patent (“Mutant Bacillus thuringiensis Cry1 genes and methods of use”; U.S. patent no. US9090906 B2 and MX339784).

Footnotes

Supplemental material is available online only.

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