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Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin
Journal of Invertebrate Pathology 114 (2013) 139–143
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Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin Dorra Ben Hamadou-Charfi a, Hanen Boukedi a, Lobna Abdelkefi-Mesrati a,⇑, Slim Tounsi a, Samir Jaoua a,b a b
Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, P.O. Box ‘1177’, 3018 Sfax, Tunisia Department of Biological & Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box ‘2713’, Doha, Qatar
a r t i c l e
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Article history: Received 21 February 2013 Accepted 12 July 2013 Available online 20 July 2013 Keywords: Bacillus thuringiensis Agrotis segetum Vip3Aa16 Cry1Ac Toxicity Midgut putative receptor
a b s t r a c t Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90 kDa) yielding three bands of about 62, 45, 22 kDa and the activated form of the toxin was active against this pest with an LC50 of about 86 ng/cm2. To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60–65 kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120 kDa protein whereas the Vip3Aa16 binds to a 65 kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis is a Gram positive bacterium with insecticidal activity due to its ability to synthesize large amounts of crystal proteins called delta-endotoxins or Cry proteins and it is the most widely used biocontrol agent (Schnepf et al., 1998). After ingestion by the insect, Cry proteins are solubilized and processed by the midgut juice to an active toxin core (Rouis et al., 2007). Active toxins bind to receptors on the brush border membrane and oligomerize to form pores that result in cell lysis, gut paralysis and larvae death (Van Rie et al., 1990). Although any variation in a step of the Cry toxin mode of action can potentially result in decreased susceptibility, alteration of toxin– receptor interactions is the most reported resistance mechanism (Ferré and Van Rie, 2002). Current ‘‘toxin-binding models’’ to explain these interactions are based on binding competition studies using radiolabeled toxins and insect midgut brush border membrane vesicles. On the basis of their toxin binding specificity, three populations of Cry1 toxin-binding sites (A, B, and C) were described in BBMV from B. thuringiensis susceptible insects (Pigott and Ellar, 2007). In addition ⇑ Corresponding author. Fax: +216 74875818. E-mail address: [email protected] (L. Abdelkefi-Mesrati). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.07.003
to Cry, several strains of B. thuringiensis are known to produce vegetative insecticidal proteins (Vip). This class of insecticidal proteins includes the binary toxin Vip1-Vip2 active on Coleoptera and Vip3 toxin active on Lepidoptera (Warren, 1997). The secreted Vip protein is structurally, functionally and biochemically different from B. thuringiensis delta-endotoxins. One of the interesting features of the Vip proteins is that they do not share sequence homology with the known delta-endotoxins (Estruch et al., 1996). Shotkoshi and Chen (2003) showed that Vip3A protein is activated by proteolysis in the lepidopteran gut and that this activation, alone, was not considered sufficient for insect specificity. In fact, ion channel analysis demonstrated that the activated Vip3A toxin must bind to specific receptors in the BBMV in order to form stable ion channels and accomplish its insecticidal activity (Lee et al., 2006). In ligand blotting experiments with BBMV from the Lepidoptera Prays oleae, activated Cry1Ac and Vip3Aa16 bound to different molecules receptors (Abdelkefi-Mesrati et al., 2009). Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelial cells resulting in larvae death at concentrations above 40 ng/cm2 (Yu et al., 1997). Compared to B. thuringiensis Cry toxins, Vip3A toxins have a mode of action that
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appears to be different especially in the binding step, and this supports its use as a novel insecticidal agent. Agrotis segetum is a pest that causes important damage on more than 50 plant crops including cereal grains (Esbjerg, 1989). BUPM95 is a B. thuringiensis subsp. kurstaki strain producing the Vip3Aa16 protoxin which is toxic against P. oleae, Spodoptera exigua, S. frugiperda, Spodoptera littoralis and Ephestia kuehniella (Abdelkefi-Mesrati et al., 2009, 2011a,b; Chakroun et al., 2012). In the present work, we studied the mode of action of Vip3Aa16 in the crop pest A. segetum in comparison with the Cry1Ac protoxin produced by the B. thuringiensis recombinant strain BNS3Cry-(pHTcry1Ac) (Tounsi et al., 2005).
2. Materials and methods 2.1. Bioassays Bioassays were carried out using first instar larvae of A. segetum. Ten larvae were transferred to sterile petri dishes containing a 1 cm2 of artificial diet. Protoxins concentrations were estimated using Bradford method (Bradford, 1976) with bovine serum albumin (BSA, Amersham) as a protein standard. To test the efficiency of Cry1Ac against A. segetum, the diet was poured with crystal/spores mixture of the recombinant B. thuringiensis strain BNS3Cry-(pHTcry1Ac), expressing the Cry1Ac d-endotoxin, previously constructed and investigated in our laboratory (Tounsi et al., 2005). Eight different concentrations (lg/cm2) of Cry1Ac proteins were tested, ten larvae were used per concentration, and each test was done in triplicate. The acrystalliferous strain BNS3Cry- was used as a negative control and mortality was recorded after 3 days at 28 °C. In the same conditions, five different concentrations (ng/cm2) of purified Vip3Aa16, as reported in Section 2.4, were poured on the surface of the diet and incubated in the presence of 10 larvae (per toxin concentration). As negative control, 10 larvae were fed with artificial diet treated with buffer solution. The mortality was recorded after 6 days at 28 °C and results are the average of three repetitions. Fifty and ninety percent lethal concentrations (LC50 and LC90) were calculated from pooled raw data by probit analysis using programs written in the R language (Venables and Smith, 2004).
2.3. Preparation of Cry1Ac toxin B. thuringiensis subsp. kurstaki BNS3Cry-(pHTcry1Ac) was grown in T3 medium (5 g peptone/1.5 g yeast extract/2 mg MnSO4 7H2O/20 mg MgSO4 7H2O/1.4 g Na2HPO4/1.2 g NaH2PO4 per liter) at 30 °C until cell lysis. The spore-crystal mixture was harvested and washed twice with 1 M NaCl, 0.01% Triton then twice with sterile water. Crystals were solubilized overnight at 37 °C in 50 mM Na2CO3, 10 mM DTT. Solubilized crystal protein was treated with trypsin (60:6-w/w) at 37 °C for 3 h. The 60kDa Cry1Ac toxin was purified by Fast protein Liquid Chromatography (FPLC; Pharmacia) using a Mono Q anion exchange column equilibrated with 20 mM Tris–HCl, pH 8. Toxin was eluted with a linear gradient of 1 M NaCl in 20 mM Tris–HCl, pH 8. Activated pure toxin was diluted in bicarbonate buffer (40 mM, pH 8.6) in order to obtain a final concentration of about 1 mg/ml. Then, 40 ll of biotinylation substrate (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) were added and the mixture was incubated at room temperature with constant agitation for 1 h. Purification of the biotinylated toxin was performed by loading the mixture on G25 column and elution using PBS (pH 7.5). 2.4. Preparation of Vip3Aa16 toxin BUPM95 is a B. thuringiensis subsp. kurstaki strain producing the Vip3Aa16 protoxin (Abdelkefi-Mesrati et al., 2005). After vip3Aa16 gene cloning, the corresponding protein was overexpressed in recombinant E. coli cells (Abdelkefi-Mesrati et al., 2009). Then, the cell pellet was suspended in PBS (pH 7.5) buffer and sonicated as described by Abdelkefi-Mesrati et al. (2009). The supernatant, collected after centrifugation and containing the Vip3Aa16 fused with six histidines, was loaded onto a His-Trap column (Amersham) preequilibrated with a binding buffer (PBS, 40 mM imidazole). After washing the column with 10 ml of the same binding buffer, the bound proteins were eluted using elution buffers containing increasing concentrations of imidazole in PBS. For binding assays, purified Vip3Aa16 protoxins were activated by proteolysis using bovine pancreas trypsin (Amersham Pharmacia Biotech, France) with a 1:40 ratio of trypsin: protoxin and incubation at 37 °C for 2 h. Activated pure toxins were treated with biotin as described above. 2.5. A. segetum BBMV preparation and competition binding assays
2.2. Preparation of whole larvae extract and proteolysis Third instar A. segetum larvae were chilled on ice during 30 min. Then, in each 1.5 ml eppendorf tube, 10 whole larvae were collected in 100 ll MET buffer 1 (300 mM Mannitol, 5 mM EDTA, 20 mM Tris pH 7.2) as described by Dammak et al. (2010). After centrifugation for 10 min at 13,000g, supernatants were recovered and the protein concentration was determined by the method of Bradford (1976), using BSA as standard. Purified Vip3Aa16 protoxins (20 lg) were mixed with soluble proteins from the larvae gut juice (3 lg) or with bovine pancreas trypsin (0.5 lg) (Amersham Pharmacia Biotech, France) in a final volume of 50 ll in PBS buffer (Phosphate buffer saline 1X). The mixtures were incubated at 37 °C with constant agitation for 1 h, 3 h, 5 h and overnight. Immunoblot analysis, with anti-Vip3Aa16 protein, was used for the proteolysis visualization. Solubilized Cry1Ac proteins (20 lg) were mixed with soluble proteins from A. segetum larvae extracts (3 lg) or with trypsin in a final volume of 50 ll. The mixtures were incubated at 37 °C for 5–180 min and overnight. Samples were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS– PAGE) and stained with Coomassie blue dye.
Midguts were dissected from fifth instar (L5) larvae, washed in ice-cold MET buffer 2 (250 mM Mannitol, 17 mM Tris–HCl, 5 mM EDTA [pH 7.5]) and kept at 80 until required. BBMV were prepared by the differential magnesium precipitation method (Wolfersberger et al., 1987) and the protein concentration was determined by the method of Bradford with BSA as a standard. For homologous competition binding assay, biotinylated trypsinized Vip3Aa16 or Cry1Ac toxins (80 nM) were incubated with BBMV (80 lg) in PBS (pH 7.6) buffer for 1 h at room temperature in the absence or presence of unlabelled trypsinized toxins (50, 200 and 400 fold). For heterologous competition binding assay, biotinylated trypsinised Cry1Ac or Vip3Aa16 toxins (80 nM) were incubated with BBMV (80 lg) in PBS (pH 7.6) buffer for 1 h at room temperature in the absence or presence of unlabeled trypsinized Vip3Aa16 or Cry1Ac toxin (200 fold), respectively. In both assays, the pellet obtained after centrifugation was suspended in 30 ll of PBS, loaded on SDS–PAGE and electrotransferred to a nitrocellulose membrane. The biotinylated proteins that were bound to BBMV were visualized by incubating with streptavidin- peroxidase conjugate (1:1500 dilutions) supplied in the
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ECL protein biotinylation module for 1 h, followed by three 5-min washing cycles. Binding was visualized using luminol according to the manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). 2.6. BBMV ligand-blotting assay BBMV (40 lg) prepared from A. segetum were separated in SDS– PAGE and blotted onto a nitrocellulose membrane by electrotransfer (Bio-Rad, France). The blots were blocked with 1% milk in PBS buffer for 1 h then reacted with biotinylated trypsinized toxins (80 nM) for 2 h at room temperature. After three 5-min washing cycles using a buffer containing PBS and 0.1% Tween 20 (pH 7.0), blots were incubated with streptavidin-peroxidase conjugate (1:1500 dilution in PBS) supplied in the ECL protein biotinylation module for 1 h, followed by three 5-min washing cycles as described above. Binding was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France).
Fig. 1. Immunoblot analysis of the activation of Vip3Aa16 using Vip3Aa16 antibody. Lane 1: unprocessed Vip3Aa16 (90 kDa); Lanes 2–5: Vip3Aa16 incubated with A. segetum proteases (3 lg) for 1 h, 3 h, 5 h and overnight, respectively.
2.7. Preparation and sectioning of insect tissues After exposure to the B. thuringiensis Vip3Aa16 protoxin, A. segetum larvae were placed in Bouin-Dubosg-Brasil solution (Picric acid, saturated aqueous solution 75 ml; Formalin, 40% aqueous solution 20 ml; Acetic acid 5 ml) for 24 h. Larvae were rinsed in 70% Ethanol and dehydrated in increasing ethanol concentrations, rinsed in pure Methylbenzoate (1 h), Benzole (30 min), BenzoleHistosec I (1v/1v for 30 min) then in Histosec I (2 h at 40 °C) and Histosec I with 0.05% DMSO (dimethyl sulfoxide) (24–48 h at 60 °C; depending on the size of the larvae), and finally embedded in paraffin wax. Nine-micrometer sections were obtained and placed in carriers coated with a mix of egg albumin and glycerol (1v/1v). For histopathological visualization of the effects of the Vip3A toxin, the 9 lm sections already de-paraffinated in 100% toluene were stained with hematoxylin eosin (HE) as reported by Ruiz et al. (2004). 3. Results and discussion 3.1. Toxicity of Vip3Aa16 and Cry1Ac against A. segetum First instar larvae of A. segetum were exposed to the purified Vip3Aa16 protoxin. The analysis of the results showed that the protein Vip3Aa16 exhibited high toxicity against A. segetum with an LC50 of 86 ng/cm2 with 95% confidence limits of (76–95 ng/cm2) and LC90 of 147 ng/cm2 with confidence limits of (126–169 ng/ cm2). The protein Cry1Ac was also toxic to A. segetum with an LC50 of 34 lg/cm2 with confidence limits of (21-47 lg/cm2) and an LC90 of 129 lg/cm2 with 95% confidence limits of (103155 ng/cm2). We noted that exposure of larvae to negative controls did not cause mortality. These results demonstrated the susceptibility of A. segetum to B. thuringiensis Vip3Aa16 and Cry1Ac proteins. Moreover, the non-killed larvae treated with Vip3Aa16 protoxin at a concentration of about 40 ng/cm2 or more were blocked at their first-instar stage. When tested against first instar larvae of E. kuehniella, Vip3Aa16 protein showed toxicity with an LC50 of 36 ng/cm2 (Abdelkefi-Mesrati et al., 2011b). Sena et al. (2009) reported that Vip3Aa is toxic to S. frugiperda with an LC50 of 49.3 ng/cm2. Against S. littoralis, Vip3Aa16 causes mortality with an LC50 and LC90 of about 305 and 1045 ng/cm2, respectively. These results demonstrated that A. segetum was 3.5 times more susceptible to Vip3Aa16 than S. littoralis, and 2.4 times less sensitive than E. kuehniella to this toxin and support the use of Vip3Aa16 in a biological control program against A. segetum.
Fig. 2. Proteolysis of Vip3Aa16 with trypsin. Lane 1: Molecular Weight Markers (97, 66, 45, 33 kDa); Lane 2: unprocessed Vip3Aa16 (90 kDa); Lane 3: Vip3Aa16 incubated with trypsin for 2 h.
3.2. Vip3 Aa16 and Cry1Ac protoxins activation Proteolysis of B. thuringiensis toxins in the midgut is a key step in determining their potency against lepidoptera since the unprocessed protein is incapable of pore forming in vitro (Lee et al., 2003). As shown in Fig. 1, Vip3Aa16 protoxin (90 kDa) was completely proteolyzed by whole larvae proteases of A. segetum after overnight incubation, yielding a major band of about 62 kDa and slight amounts of two other bands of about 45 and 22 kDa (Fig. 1, Lane 5); whereas the activation of Vip3Aa16 with trypsin yields a stable toxin of 62 kDa (Fig. 2, Lane 3). The activation of Vip3Aa16 with A. segetum gut proteases is in agreement with previous reports. Yu et al. (1997) demonstrated that Vip3A can be processed into four major proteolysis products of approximately
Fig. 3. Activation of the protoxin Cry1Ac. Lane 1: Molecular Weight Markers (150, 100, 70, 50, 40, 30 kDa); Lane 2: unprocessed protoxin Cry1Ac; Lanes 3–7: Cry1Ac (130 kDa) incubated with A. segetum proteases for 5 min, 30 min, 1 h, 3 h and overnight, respectively; Lane 8: Cry1Ac incubated with trypsin.
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62, 45, 33, and 22 kDa by lepidopteran gut fluids. The Vip3Aa16 protoxin is activated with S. littoralis gut fluids yielding four proteolysis products of 62, 45, 33 and 22 kDa (Abdelkefi-Mesrati et al., 2011a). In the case of E. kuehniella, this protoxin is activated to a major band of 62 kDa and another band of 45 kDa (Abdelkefi-Mesrati et al., 2011b). Lee et al. (2003) demonstrated that a dominant stable 62 kDa protein is formed by the action of lepidopteran gut juice extract on the protoxin (90 kDa) and that this 62 kDa band corresponds to the active form of the Vip3A toxin. But alone, protoxin activation is not a determining factor for insect specificity. In fact, Vip3A protoxin can be processed by the action of gut juice from the non susceptible insect, Ostrinia nubilalis (Yu et al., 1997). The whole larvae proteases of A. segetum activated differently the Cry1Ac protoxin. Before incubation with whole larvae protease extract, partially purified Cry1Ac protoxins showed, after SDS– PAGE, a major protein of 130 kDa (Fig. 3, Lane 2). The proteolysis of the protoxin (130 kDa) increases with time between 5 and 180 min until complete activation after overnight incubation with the larvae midgut proteases. In fact, Cry1Ac protoxin (130 kDa) can be activated with whole A. segetum protease extract to a triplet of 60–65 kDa (Fig. 3, Lane 7). The proteolysis with trypsin was slightly different from gut proteases yielding a major band of 65 kDa (Fig. 3, Lane 8). Similar results were reported by Rouis et al. (2007) regarding the Cry1Ac protoxin activation with E. kuehniella midgut proteases.
recognized by Cry1Ac (210 kDa) (Abdelkefi-Mesrati et al., 2009). Whereas in S. littoralis, Vip3Aa16 toxin bound to two putative receptors of about 55 and 100 kDa (Abdelkefi-Mesrati et al., 2011a). In Manduca sexta, biotinylated gut juice activated Vip3A toxin predominantly binds to 80 and 110 kDa bands, generating a pattern that clearly differs from that of Cry1Ab (120 kDa) (Lee et al., 2003). In the case of A. segetum, the differences between the putative receptors recognized by Vip3Aa16 and Cry1Ac toxins supports the use of Vip3 toxins as a biological control agent, especially to resolve the problems of Cry-resistance emergence.
3.3. Vip3Aa16 and Cry1Ac binding to BBMV
3.4. BBMV competition binding properties
BBMV ligand blotting assays were performed to identify putative receptors of B. thuringiensis toxins on BBMV proteins. Previously, several putative Cry1Ac toxin-binding proteins in Heliothis virescens have been reported (80, 120, 150, 170, and >210 kDa) using BBMV ligand blotting technique (Lee et al., 1995; Luo et al., 1997; Banks et al., 2001). In the present study, biotinylated trypsinized Cry1Ac binds to a putative receptor of about 120 kDa (Fig. 4A, Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated trypsinized Cry1Ac toxin of B. thuringiensis (Fig. 4A, Lane 2). After BBMV proteins electro- transferred to a nitrocellulose membrane, biotinylated trypsinized Vip3Aa16 toxin bound to one putative receptor of about 65 kDa (Fig. 4B, Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated trypsinized Vip3Aa16 toxin of B. thuringiensis (Fig. 4B, Lane 2). Similar results are described by Abdelkefi-Mesrati et al. (2011b) regarding the 65 kDa putative receptor of Vip3Aa16 toxin on E. kuehniella BBMV. In P. oleae midgut, Vip3Aa16 toxin recognizes a putative binding receptor of about 65 kDa which differs from that
One of the key elements in the mode of action of B. thuringiensis toxin is its binding to specific receptor(s) on the surface of the midgut epithelial brush border membrane. Homologous BBMV competition binding assays with A. segetum were performed. Vip3Aa16 and Cry1Ac toxin-binding properties have been assessed by competition binding assays using biotinylated trypsinized Vip3Aa16 and Cry1Ac, respectively. Homologous competition experiments were done by incubating the labeled toxin in the presence or the absence of unlabelled toxin and the appropriate BBMV concentration. As shown in Fig. 5A and B, Cry1Ac and Vip3Aa16 bound specifically since an increasing amount of the same unlabelled protein significantly reduced the binding of the labeled protein. Similar results were obtained with Vip3Af and BBMV of S. frugiperda (Sena et al., 2009) and with Vip3Aa16 and BBMV of S. littoralis (Abdelkefi-Mesrati et al., 2011a). To study the binding sites relationship between Vip3Aa16 and Cry1Ac, heterologous competition binding assays were performed with incubation of the biotinylated Cry1Ac toxin in the absence or presence of the unlabeled Vip3Aa16 toxin (200 fold) and the appropriate BBMV concentration. No competitive binding was observed between Vip3A and Cry1Ac toxins. In fact, excess amounts of cold Vip3Aa16 toxin (200 fold) did not inhibit the binding of the labeled Cry1Ac toxin (Fig. 6A, Lane 2), indicating that Vip3Aa16 does not bind to the Cry1Ac binding sites. Similar results were obtained when biotinylated Vip3Aa16 toxin was incubated in the absence or presence of the unlabeled Cry1Ac toxin (200 fold) and the appropriate BBMV concentration demonstrating that Cry1Ac does not bind to the Vip3Aa16 binding sites (Fig. 6B, Lane 2). These specific binding properties have also been previously demonstrated by
Fig. 4. Cry1Ac and Vip3Aa16 putative receptor in A. segetum midgut. (A) A. segetum putative receptor of Cry1Ac toxin: Lane 1, A. segetum BBMV ligand blotting with biotinylated Cry1Ac toxin. Lane 2, containing bovine serum albumin (BSA) was used as negative control. (B) A. segetum putative receptor of Vip3Aa16 toxin: Lane 1, A. segetum BBMV ligand blotting with biotinylated Vip3Aa16 toxin. Lane 2, containing BSA was used as negative control.
Fig. 5. Homologous competition binding assays on A. segetum BBMV: (A) biotinylated trypsin-activated Cry1Ac toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of an excess of unlabelled Cry1Ac toxin (Lane 2, 50X, Lane 3, 200X, Lane 4, 400X). (B) Biotinylated trypsin-activated Vip3Aa16 toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of an excess of unlabelled Vip3Aa16 toxin (Lane 2, 50X, Lane 3, 200X, Lane 4, 400X).
Fig. 6. Heterologous competition binding assays on A. segetum BBMV. (A) Biotinylated trypsin-activated Cry1Ac toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of unlabelled Vip3Aa16 toxin (200X; Lane 2). (B) Biotinylated trypsin-activated Vip3Aa16 toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of unlabelled Cry1Ac toxin (200X; Lane 2).
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Fig. 7. Histopathological effects of Vip3Aa16 on A. segetum midgut: general aspects of the midgut larvae (A) and histopathological effects of Vip3Aa16 on it (B). B: a strong vacuolization of columnar cells, white arrows indicate lysis of columnar cells. Lu, lumen; Gc, goblet cell, Am, apical membrane, Bm, basal membrane. Magnification 40X.
Lee et al. (2006) with Vip3A and Cry1Ac in the BBMV of H. virescens and Helicoverpa zea. In the present study, we demonstrated that Vip3Aa16 does not interfere with the binding sites of Cry1Ac. These distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum particularly in case of resistance emergence to Cry proteins. 3.5. Histopathological effect of the Vip3Aa16 toxin in A. segetum larvae Due to its important toxicity against A. segetum, the histopathological effect of Vip3Aa16 on these larvae was studied. First instars larvae had been fed on diet containing the protoxin. In the control tissue, the midgut of untreated A. segetum larvae showed uniform morphology and well-defined epithelial cells with unaffected apical microvilli membrane (Fig. 7A). As shown in Fig. 7B, extensive damage was detected in the midgut epithelium, indicating that the midgut tissue is a primary site of action of the Vip3Aa16 toxin. The histopathological modifications included vacuolization of the cytoplasm, brush border membrane lysis and disintegration of the apical membrane. The present study demonstrated the potential of Vip3Aa16 against the crop pest A. segetum. The differences in the binding properties between Vip3 and Cry1Ac toxins enhance the use of Vip3Aa16 as a novel bioinsecticidal agent against the polyphagous A. segetum especially in the case of Cryresistance development in this pest. Acknowledgments This work was supported by Grants from the ‘‘Ministère de l’Enseignement Supérieur et de la Recherche Scientifique’’. We thank Dr. Souad Rouis and Mrs Dalel Ben farhat for their assistance during this study. References Abdelkefi-Mesrati, L., Tounsi, S., Jaoua, S., 2005. Characterization of a novel vip3type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol. Lett. 244, 353–358. Abdelkefi-Mesrati, L., Rouis, S., Sellami, S., Jaoua, S., 2009. Prays oleae midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3LB differs from that of Cry1Ac toxin. Mol. Biotechnol. 43, 15–19. Abdelkefi-Mesrati, L., Boukedi, H., Dammak-Karray, M., Sellami-Boudawara, T., Jaoua, S., Tounsi, S., 2011a. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. J. Invertebr. Pathol. 106, 250–254. Abdelkefi-Mesrati, L., Boukedi, H., Chakroun, M., Kamoun, F., Azzouz, H., Tounsi, S., Rouis, S., Jaoua, S., 2011b. Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin. J. Invertebr. Pathol. 107, 198–201. Banks, D.J., Jurat-Fuentes, J.L., Dean, D.H., Adang, M.J., 2001. Bacillus thuringiensis Cry1Ac and Cry1Fa d-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated. Insect Biochem. Mol. Biol. 31, 909–918.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chakroun, M., Bel, Y., Caccia, S., Abdelkefi-Mesrati, L., Escriche, B., Ferré, J., 2012. Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein. J. Invertebr. Pathol. 110, 334–339. Dammak, M., Tounsi, S., Rouis, S., Jaoua, S., 2010. Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: activation process and toxicity against Prays oleae. J. Invertebr. Pathol. 104, 39–43. Esbjerg, P.H., 1989. The influence of soil moisture on mortality and on the damaging effect of 2nd to 6th instar cutworms (Agrotis segetum Schiff, Lep.: Nctuidae. Ecol. Applic. 10, 335–347. Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc. Natl. Acad. Sci. USA 93, 5389–5394. Ferré, J., Van Rie, J., 2002. Biochemistry and genetics of insects resistance to Bacillus thuringiensis. Ann. Rev. Entomol. 47, 501–533. Lee, M.K., Rajamohan, F., Gould, F., Dean, D.H., 1995. Resistance to Bacillus thuringiensis Cry1A d-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration. Appl. Environ. Microbiol. 61, 3836–3842. Lee, M.K., Walters, F.S., Hart, H., Palekar, N., Chen, J.S., 2003. The mode of action of the Bacillus thuringiensis Vegetative Insecticidal Protein VIP3A differs from that of Cry1Ab d-endotoxin. Appl. Environ. Microbiol. 69, 4648–4657. Lee, M.K., Miles, P., Chen, J.S., 2006. Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem. Biophys. Res. Commun. 339, 1043–1047. Luo, K., Sangadala, S., Masson, L., Mazza, A., Brousseau, R., Adang, M.J., 1997. The Heliothis virescens 170 kDa aminopeptidase functions as ‘‘receptor A’’ by mediating specific Bacillus thuringiensis d-endotoxin binding and pore formation. Insect Biochem. Mol. Biol. 27, 735–743. Pigott, C.R., Ellar, D.J., 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Mol. Biol. Rev. 71, 255–281. Rouis, S., Chakroun, M., Saadaoui, I., Jaoua, S., 2007. Proteolysis, histopathological effects, and immunohistopathological localization of d-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of Lepidopteran olive tree pathogenic insect Prays oleae. Mol. Biotechnol. 35, 141–148. Ruiz, L.M., Segura, C., Trujillo, J., Orduz, S., 2004. In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz. 99, 73–79. Sena, J.A.D., Hernandez-Rodríguez, C.S., Ferré, J., 2009. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites. Appl. Environ. Microbiol. 75, 2236–2237. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Shotkoshi, F., Chen, F., 2003. Vip: a novel insecticidal protein with broad spectrum Lepidopteran activity. In: Proc. Belwide Cotton Conf., Nashville TN. vol. 6, pp. 89–93. Tounsi, S., Dammak, M., Rebai, A., Jaoua, S., 2005. Response of larval Ephestia kuehniella (Lepidoptera/Pyralidae) to individual Bacillus thuringiensis kurstaki toxins and toxin mixtures. Biol. Control 35, 27–31. Van Rie, J., McGaughey, W.H., Johnson, D.E., Barnett, D.B., Van Mellaert, H., 1990. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science 247, 72–74. Venables, W.N., Smith, D.M., 2004. The R. development core team. An introduction to R. version 1.9.1.
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin Dorra Ben Hamadou-Charfi a, Hanen Boukedi a, Lobna Abdelkefi-Mesrati a,⇑, Slim Tounsi a, Samir Jaoua a,b a b
Biopesticides Team (LPAP), Centre of Biotechnology of Sfax, P.O. Box ‘1177’, 3018 Sfax, Tunisia Department of Biological & Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box ‘2713’, Doha, Qatar
a r t i c l e
i n f o
Article history: Received 21 February 2013 Accepted 12 July 2013 Available online 20 July 2013 Keywords: Bacillus thuringiensis Agrotis segetum Vip3Aa16 Cry1Ac Toxicity Midgut putative receptor
a b s t r a c t Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90 kDa) yielding three bands of about 62, 45, 22 kDa and the activated form of the toxin was active against this pest with an LC50 of about 86 ng/cm2. To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60–65 kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120 kDa protein whereas the Vip3Aa16 binds to a 65 kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bacillus thuringiensis is a Gram positive bacterium with insecticidal activity due to its ability to synthesize large amounts of crystal proteins called delta-endotoxins or Cry proteins and it is the most widely used biocontrol agent (Schnepf et al., 1998). After ingestion by the insect, Cry proteins are solubilized and processed by the midgut juice to an active toxin core (Rouis et al., 2007). Active toxins bind to receptors on the brush border membrane and oligomerize to form pores that result in cell lysis, gut paralysis and larvae death (Van Rie et al., 1990). Although any variation in a step of the Cry toxin mode of action can potentially result in decreased susceptibility, alteration of toxin– receptor interactions is the most reported resistance mechanism (Ferré and Van Rie, 2002). Current ‘‘toxin-binding models’’ to explain these interactions are based on binding competition studies using radiolabeled toxins and insect midgut brush border membrane vesicles. On the basis of their toxin binding specificity, three populations of Cry1 toxin-binding sites (A, B, and C) were described in BBMV from B. thuringiensis susceptible insects (Pigott and Ellar, 2007). In addition ⇑ Corresponding author. Fax: +216 74875818. E-mail address: [email protected] (L. Abdelkefi-Mesrati). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.07.003
to Cry, several strains of B. thuringiensis are known to produce vegetative insecticidal proteins (Vip). This class of insecticidal proteins includes the binary toxin Vip1-Vip2 active on Coleoptera and Vip3 toxin active on Lepidoptera (Warren, 1997). The secreted Vip protein is structurally, functionally and biochemically different from B. thuringiensis delta-endotoxins. One of the interesting features of the Vip proteins is that they do not share sequence homology with the known delta-endotoxins (Estruch et al., 1996). Shotkoshi and Chen (2003) showed that Vip3A protein is activated by proteolysis in the lepidopteran gut and that this activation, alone, was not considered sufficient for insect specificity. In fact, ion channel analysis demonstrated that the activated Vip3A toxin must bind to specific receptors in the BBMV in order to form stable ion channels and accomplish its insecticidal activity (Lee et al., 2006). In ligand blotting experiments with BBMV from the Lepidoptera Prays oleae, activated Cry1Ac and Vip3Aa16 bound to different molecules receptors (Abdelkefi-Mesrati et al., 2009). Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelial cells resulting in larvae death at concentrations above 40 ng/cm2 (Yu et al., 1997). Compared to B. thuringiensis Cry toxins, Vip3A toxins have a mode of action that
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appears to be different especially in the binding step, and this supports its use as a novel insecticidal agent. Agrotis segetum is a pest that causes important damage on more than 50 plant crops including cereal grains (Esbjerg, 1989). BUPM95 is a B. thuringiensis subsp. kurstaki strain producing the Vip3Aa16 protoxin which is toxic against P. oleae, Spodoptera exigua, S. frugiperda, Spodoptera littoralis and Ephestia kuehniella (Abdelkefi-Mesrati et al., 2009, 2011a,b; Chakroun et al., 2012). In the present work, we studied the mode of action of Vip3Aa16 in the crop pest A. segetum in comparison with the Cry1Ac protoxin produced by the B. thuringiensis recombinant strain BNS3Cry-(pHTcry1Ac) (Tounsi et al., 2005).
2. Materials and methods 2.1. Bioassays Bioassays were carried out using first instar larvae of A. segetum. Ten larvae were transferred to sterile petri dishes containing a 1 cm2 of artificial diet. Protoxins concentrations were estimated using Bradford method (Bradford, 1976) with bovine serum albumin (BSA, Amersham) as a protein standard. To test the efficiency of Cry1Ac against A. segetum, the diet was poured with crystal/spores mixture of the recombinant B. thuringiensis strain BNS3Cry-(pHTcry1Ac), expressing the Cry1Ac d-endotoxin, previously constructed and investigated in our laboratory (Tounsi et al., 2005). Eight different concentrations (lg/cm2) of Cry1Ac proteins were tested, ten larvae were used per concentration, and each test was done in triplicate. The acrystalliferous strain BNS3Cry- was used as a negative control and mortality was recorded after 3 days at 28 °C. In the same conditions, five different concentrations (ng/cm2) of purified Vip3Aa16, as reported in Section 2.4, were poured on the surface of the diet and incubated in the presence of 10 larvae (per toxin concentration). As negative control, 10 larvae were fed with artificial diet treated with buffer solution. The mortality was recorded after 6 days at 28 °C and results are the average of three repetitions. Fifty and ninety percent lethal concentrations (LC50 and LC90) were calculated from pooled raw data by probit analysis using programs written in the R language (Venables and Smith, 2004).
2.3. Preparation of Cry1Ac toxin B. thuringiensis subsp. kurstaki BNS3Cry-(pHTcry1Ac) was grown in T3 medium (5 g peptone/1.5 g yeast extract/2 mg MnSO4 7H2O/20 mg MgSO4 7H2O/1.4 g Na2HPO4/1.2 g NaH2PO4 per liter) at 30 °C until cell lysis. The spore-crystal mixture was harvested and washed twice with 1 M NaCl, 0.01% Triton then twice with sterile water. Crystals were solubilized overnight at 37 °C in 50 mM Na2CO3, 10 mM DTT. Solubilized crystal protein was treated with trypsin (60:6-w/w) at 37 °C for 3 h. The 60kDa Cry1Ac toxin was purified by Fast protein Liquid Chromatography (FPLC; Pharmacia) using a Mono Q anion exchange column equilibrated with 20 mM Tris–HCl, pH 8. Toxin was eluted with a linear gradient of 1 M NaCl in 20 mM Tris–HCl, pH 8. Activated pure toxin was diluted in bicarbonate buffer (40 mM, pH 8.6) in order to obtain a final concentration of about 1 mg/ml. Then, 40 ll of biotinylation substrate (ECL™ protein biotinylation module: Amersham Pharmacia Biotech, France) were added and the mixture was incubated at room temperature with constant agitation for 1 h. Purification of the biotinylated toxin was performed by loading the mixture on G25 column and elution using PBS (pH 7.5). 2.4. Preparation of Vip3Aa16 toxin BUPM95 is a B. thuringiensis subsp. kurstaki strain producing the Vip3Aa16 protoxin (Abdelkefi-Mesrati et al., 2005). After vip3Aa16 gene cloning, the corresponding protein was overexpressed in recombinant E. coli cells (Abdelkefi-Mesrati et al., 2009). Then, the cell pellet was suspended in PBS (pH 7.5) buffer and sonicated as described by Abdelkefi-Mesrati et al. (2009). The supernatant, collected after centrifugation and containing the Vip3Aa16 fused with six histidines, was loaded onto a His-Trap column (Amersham) preequilibrated with a binding buffer (PBS, 40 mM imidazole). After washing the column with 10 ml of the same binding buffer, the bound proteins were eluted using elution buffers containing increasing concentrations of imidazole in PBS. For binding assays, purified Vip3Aa16 protoxins were activated by proteolysis using bovine pancreas trypsin (Amersham Pharmacia Biotech, France) with a 1:40 ratio of trypsin: protoxin and incubation at 37 °C for 2 h. Activated pure toxins were treated with biotin as described above. 2.5. A. segetum BBMV preparation and competition binding assays
2.2. Preparation of whole larvae extract and proteolysis Third instar A. segetum larvae were chilled on ice during 30 min. Then, in each 1.5 ml eppendorf tube, 10 whole larvae were collected in 100 ll MET buffer 1 (300 mM Mannitol, 5 mM EDTA, 20 mM Tris pH 7.2) as described by Dammak et al. (2010). After centrifugation for 10 min at 13,000g, supernatants were recovered and the protein concentration was determined by the method of Bradford (1976), using BSA as standard. Purified Vip3Aa16 protoxins (20 lg) were mixed with soluble proteins from the larvae gut juice (3 lg) or with bovine pancreas trypsin (0.5 lg) (Amersham Pharmacia Biotech, France) in a final volume of 50 ll in PBS buffer (Phosphate buffer saline 1X). The mixtures were incubated at 37 °C with constant agitation for 1 h, 3 h, 5 h and overnight. Immunoblot analysis, with anti-Vip3Aa16 protein, was used for the proteolysis visualization. Solubilized Cry1Ac proteins (20 lg) were mixed with soluble proteins from A. segetum larvae extracts (3 lg) or with trypsin in a final volume of 50 ll. The mixtures were incubated at 37 °C for 5–180 min and overnight. Samples were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS– PAGE) and stained with Coomassie blue dye.
Midguts were dissected from fifth instar (L5) larvae, washed in ice-cold MET buffer 2 (250 mM Mannitol, 17 mM Tris–HCl, 5 mM EDTA [pH 7.5]) and kept at 80 until required. BBMV were prepared by the differential magnesium precipitation method (Wolfersberger et al., 1987) and the protein concentration was determined by the method of Bradford with BSA as a standard. For homologous competition binding assay, biotinylated trypsinized Vip3Aa16 or Cry1Ac toxins (80 nM) were incubated with BBMV (80 lg) in PBS (pH 7.6) buffer for 1 h at room temperature in the absence or presence of unlabelled trypsinized toxins (50, 200 and 400 fold). For heterologous competition binding assay, biotinylated trypsinised Cry1Ac or Vip3Aa16 toxins (80 nM) were incubated with BBMV (80 lg) in PBS (pH 7.6) buffer for 1 h at room temperature in the absence or presence of unlabeled trypsinized Vip3Aa16 or Cry1Ac toxin (200 fold), respectively. In both assays, the pellet obtained after centrifugation was suspended in 30 ll of PBS, loaded on SDS–PAGE and electrotransferred to a nitrocellulose membrane. The biotinylated proteins that were bound to BBMV were visualized by incubating with streptavidin- peroxidase conjugate (1:1500 dilutions) supplied in the
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ECL protein biotinylation module for 1 h, followed by three 5-min washing cycles. Binding was visualized using luminol according to the manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France). 2.6. BBMV ligand-blotting assay BBMV (40 lg) prepared from A. segetum were separated in SDS– PAGE and blotted onto a nitrocellulose membrane by electrotransfer (Bio-Rad, France). The blots were blocked with 1% milk in PBS buffer for 1 h then reacted with biotinylated trypsinized toxins (80 nM) for 2 h at room temperature. After three 5-min washing cycles using a buffer containing PBS and 0.1% Tween 20 (pH 7.0), blots were incubated with streptavidin-peroxidase conjugate (1:1500 dilution in PBS) supplied in the ECL protein biotinylation module for 1 h, followed by three 5-min washing cycles as described above. Binding was visualized using luminol according to manufacturer’s protocol (ECL; Amersham Pharmacia Biotech, France).
Fig. 1. Immunoblot analysis of the activation of Vip3Aa16 using Vip3Aa16 antibody. Lane 1: unprocessed Vip3Aa16 (90 kDa); Lanes 2–5: Vip3Aa16 incubated with A. segetum proteases (3 lg) for 1 h, 3 h, 5 h and overnight, respectively.
2.7. Preparation and sectioning of insect tissues After exposure to the B. thuringiensis Vip3Aa16 protoxin, A. segetum larvae were placed in Bouin-Dubosg-Brasil solution (Picric acid, saturated aqueous solution 75 ml; Formalin, 40% aqueous solution 20 ml; Acetic acid 5 ml) for 24 h. Larvae were rinsed in 70% Ethanol and dehydrated in increasing ethanol concentrations, rinsed in pure Methylbenzoate (1 h), Benzole (30 min), BenzoleHistosec I (1v/1v for 30 min) then in Histosec I (2 h at 40 °C) and Histosec I with 0.05% DMSO (dimethyl sulfoxide) (24–48 h at 60 °C; depending on the size of the larvae), and finally embedded in paraffin wax. Nine-micrometer sections were obtained and placed in carriers coated with a mix of egg albumin and glycerol (1v/1v). For histopathological visualization of the effects of the Vip3A toxin, the 9 lm sections already de-paraffinated in 100% toluene were stained with hematoxylin eosin (HE) as reported by Ruiz et al. (2004). 3. Results and discussion 3.1. Toxicity of Vip3Aa16 and Cry1Ac against A. segetum First instar larvae of A. segetum were exposed to the purified Vip3Aa16 protoxin. The analysis of the results showed that the protein Vip3Aa16 exhibited high toxicity against A. segetum with an LC50 of 86 ng/cm2 with 95% confidence limits of (76–95 ng/cm2) and LC90 of 147 ng/cm2 with confidence limits of (126–169 ng/ cm2). The protein Cry1Ac was also toxic to A. segetum with an LC50 of 34 lg/cm2 with confidence limits of (21-47 lg/cm2) and an LC90 of 129 lg/cm2 with 95% confidence limits of (103155 ng/cm2). We noted that exposure of larvae to negative controls did not cause mortality. These results demonstrated the susceptibility of A. segetum to B. thuringiensis Vip3Aa16 and Cry1Ac proteins. Moreover, the non-killed larvae treated with Vip3Aa16 protoxin at a concentration of about 40 ng/cm2 or more were blocked at their first-instar stage. When tested against first instar larvae of E. kuehniella, Vip3Aa16 protein showed toxicity with an LC50 of 36 ng/cm2 (Abdelkefi-Mesrati et al., 2011b). Sena et al. (2009) reported that Vip3Aa is toxic to S. frugiperda with an LC50 of 49.3 ng/cm2. Against S. littoralis, Vip3Aa16 causes mortality with an LC50 and LC90 of about 305 and 1045 ng/cm2, respectively. These results demonstrated that A. segetum was 3.5 times more susceptible to Vip3Aa16 than S. littoralis, and 2.4 times less sensitive than E. kuehniella to this toxin and support the use of Vip3Aa16 in a biological control program against A. segetum.
Fig. 2. Proteolysis of Vip3Aa16 with trypsin. Lane 1: Molecular Weight Markers (97, 66, 45, 33 kDa); Lane 2: unprocessed Vip3Aa16 (90 kDa); Lane 3: Vip3Aa16 incubated with trypsin for 2 h.
3.2. Vip3 Aa16 and Cry1Ac protoxins activation Proteolysis of B. thuringiensis toxins in the midgut is a key step in determining their potency against lepidoptera since the unprocessed protein is incapable of pore forming in vitro (Lee et al., 2003). As shown in Fig. 1, Vip3Aa16 protoxin (90 kDa) was completely proteolyzed by whole larvae proteases of A. segetum after overnight incubation, yielding a major band of about 62 kDa and slight amounts of two other bands of about 45 and 22 kDa (Fig. 1, Lane 5); whereas the activation of Vip3Aa16 with trypsin yields a stable toxin of 62 kDa (Fig. 2, Lane 3). The activation of Vip3Aa16 with A. segetum gut proteases is in agreement with previous reports. Yu et al. (1997) demonstrated that Vip3A can be processed into four major proteolysis products of approximately
Fig. 3. Activation of the protoxin Cry1Ac. Lane 1: Molecular Weight Markers (150, 100, 70, 50, 40, 30 kDa); Lane 2: unprocessed protoxin Cry1Ac; Lanes 3–7: Cry1Ac (130 kDa) incubated with A. segetum proteases for 5 min, 30 min, 1 h, 3 h and overnight, respectively; Lane 8: Cry1Ac incubated with trypsin.
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62, 45, 33, and 22 kDa by lepidopteran gut fluids. The Vip3Aa16 protoxin is activated with S. littoralis gut fluids yielding four proteolysis products of 62, 45, 33 and 22 kDa (Abdelkefi-Mesrati et al., 2011a). In the case of E. kuehniella, this protoxin is activated to a major band of 62 kDa and another band of 45 kDa (Abdelkefi-Mesrati et al., 2011b). Lee et al. (2003) demonstrated that a dominant stable 62 kDa protein is formed by the action of lepidopteran gut juice extract on the protoxin (90 kDa) and that this 62 kDa band corresponds to the active form of the Vip3A toxin. But alone, protoxin activation is not a determining factor for insect specificity. In fact, Vip3A protoxin can be processed by the action of gut juice from the non susceptible insect, Ostrinia nubilalis (Yu et al., 1997). The whole larvae proteases of A. segetum activated differently the Cry1Ac protoxin. Before incubation with whole larvae protease extract, partially purified Cry1Ac protoxins showed, after SDS– PAGE, a major protein of 130 kDa (Fig. 3, Lane 2). The proteolysis of the protoxin (130 kDa) increases with time between 5 and 180 min until complete activation after overnight incubation with the larvae midgut proteases. In fact, Cry1Ac protoxin (130 kDa) can be activated with whole A. segetum protease extract to a triplet of 60–65 kDa (Fig. 3, Lane 7). The proteolysis with trypsin was slightly different from gut proteases yielding a major band of 65 kDa (Fig. 3, Lane 8). Similar results were reported by Rouis et al. (2007) regarding the Cry1Ac protoxin activation with E. kuehniella midgut proteases.
recognized by Cry1Ac (210 kDa) (Abdelkefi-Mesrati et al., 2009). Whereas in S. littoralis, Vip3Aa16 toxin bound to two putative receptors of about 55 and 100 kDa (Abdelkefi-Mesrati et al., 2011a). In Manduca sexta, biotinylated gut juice activated Vip3A toxin predominantly binds to 80 and 110 kDa bands, generating a pattern that clearly differs from that of Cry1Ab (120 kDa) (Lee et al., 2003). In the case of A. segetum, the differences between the putative receptors recognized by Vip3Aa16 and Cry1Ac toxins supports the use of Vip3 toxins as a biological control agent, especially to resolve the problems of Cry-resistance emergence.
3.3. Vip3Aa16 and Cry1Ac binding to BBMV
3.4. BBMV competition binding properties
BBMV ligand blotting assays were performed to identify putative receptors of B. thuringiensis toxins on BBMV proteins. Previously, several putative Cry1Ac toxin-binding proteins in Heliothis virescens have been reported (80, 120, 150, 170, and >210 kDa) using BBMV ligand blotting technique (Lee et al., 1995; Luo et al., 1997; Banks et al., 2001). In the present study, biotinylated trypsinized Cry1Ac binds to a putative receptor of about 120 kDa (Fig. 4A, Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated trypsinized Cry1Ac toxin of B. thuringiensis (Fig. 4A, Lane 2). After BBMV proteins electro- transferred to a nitrocellulose membrane, biotinylated trypsinized Vip3Aa16 toxin bound to one putative receptor of about 65 kDa (Fig. 4B, Lane 1). A BSA blot, used as negative control, showed no interaction with the biotinylated trypsinized Vip3Aa16 toxin of B. thuringiensis (Fig. 4B, Lane 2). Similar results are described by Abdelkefi-Mesrati et al. (2011b) regarding the 65 kDa putative receptor of Vip3Aa16 toxin on E. kuehniella BBMV. In P. oleae midgut, Vip3Aa16 toxin recognizes a putative binding receptor of about 65 kDa which differs from that
One of the key elements in the mode of action of B. thuringiensis toxin is its binding to specific receptor(s) on the surface of the midgut epithelial brush border membrane. Homologous BBMV competition binding assays with A. segetum were performed. Vip3Aa16 and Cry1Ac toxin-binding properties have been assessed by competition binding assays using biotinylated trypsinized Vip3Aa16 and Cry1Ac, respectively. Homologous competition experiments were done by incubating the labeled toxin in the presence or the absence of unlabelled toxin and the appropriate BBMV concentration. As shown in Fig. 5A and B, Cry1Ac and Vip3Aa16 bound specifically since an increasing amount of the same unlabelled protein significantly reduced the binding of the labeled protein. Similar results were obtained with Vip3Af and BBMV of S. frugiperda (Sena et al., 2009) and with Vip3Aa16 and BBMV of S. littoralis (Abdelkefi-Mesrati et al., 2011a). To study the binding sites relationship between Vip3Aa16 and Cry1Ac, heterologous competition binding assays were performed with incubation of the biotinylated Cry1Ac toxin in the absence or presence of the unlabeled Vip3Aa16 toxin (200 fold) and the appropriate BBMV concentration. No competitive binding was observed between Vip3A and Cry1Ac toxins. In fact, excess amounts of cold Vip3Aa16 toxin (200 fold) did not inhibit the binding of the labeled Cry1Ac toxin (Fig. 6A, Lane 2), indicating that Vip3Aa16 does not bind to the Cry1Ac binding sites. Similar results were obtained when biotinylated Vip3Aa16 toxin was incubated in the absence or presence of the unlabeled Cry1Ac toxin (200 fold) and the appropriate BBMV concentration demonstrating that Cry1Ac does not bind to the Vip3Aa16 binding sites (Fig. 6B, Lane 2). These specific binding properties have also been previously demonstrated by
Fig. 4. Cry1Ac and Vip3Aa16 putative receptor in A. segetum midgut. (A) A. segetum putative receptor of Cry1Ac toxin: Lane 1, A. segetum BBMV ligand blotting with biotinylated Cry1Ac toxin. Lane 2, containing bovine serum albumin (BSA) was used as negative control. (B) A. segetum putative receptor of Vip3Aa16 toxin: Lane 1, A. segetum BBMV ligand blotting with biotinylated Vip3Aa16 toxin. Lane 2, containing BSA was used as negative control.
Fig. 5. Homologous competition binding assays on A. segetum BBMV: (A) biotinylated trypsin-activated Cry1Ac toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of an excess of unlabelled Cry1Ac toxin (Lane 2, 50X, Lane 3, 200X, Lane 4, 400X). (B) Biotinylated trypsin-activated Vip3Aa16 toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of an excess of unlabelled Vip3Aa16 toxin (Lane 2, 50X, Lane 3, 200X, Lane 4, 400X).
Fig. 6. Heterologous competition binding assays on A. segetum BBMV. (A) Biotinylated trypsin-activated Cry1Ac toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of unlabelled Vip3Aa16 toxin (200X; Lane 2). (B) Biotinylated trypsin-activated Vip3Aa16 toxin was incubated with the BBMV in the absence (Lane 1) or in the presence of unlabelled Cry1Ac toxin (200X; Lane 2).
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Fig. 7. Histopathological effects of Vip3Aa16 on A. segetum midgut: general aspects of the midgut larvae (A) and histopathological effects of Vip3Aa16 on it (B). B: a strong vacuolization of columnar cells, white arrows indicate lysis of columnar cells. Lu, lumen; Gc, goblet cell, Am, apical membrane, Bm, basal membrane. Magnification 40X.
Lee et al. (2006) with Vip3A and Cry1Ac in the BBMV of H. virescens and Helicoverpa zea. In the present study, we demonstrated that Vip3Aa16 does not interfere with the binding sites of Cry1Ac. These distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum particularly in case of resistance emergence to Cry proteins. 3.5. Histopathological effect of the Vip3Aa16 toxin in A. segetum larvae Due to its important toxicity against A. segetum, the histopathological effect of Vip3Aa16 on these larvae was studied. First instars larvae had been fed on diet containing the protoxin. In the control tissue, the midgut of untreated A. segetum larvae showed uniform morphology and well-defined epithelial cells with unaffected apical microvilli membrane (Fig. 7A). As shown in Fig. 7B, extensive damage was detected in the midgut epithelium, indicating that the midgut tissue is a primary site of action of the Vip3Aa16 toxin. The histopathological modifications included vacuolization of the cytoplasm, brush border membrane lysis and disintegration of the apical membrane. The present study demonstrated the potential of Vip3Aa16 against the crop pest A. segetum. The differences in the binding properties between Vip3 and Cry1Ac toxins enhance the use of Vip3Aa16 as a novel bioinsecticidal agent against the polyphagous A. segetum especially in the case of Cryresistance development in this pest. Acknowledgments This work was supported by Grants from the ‘‘Ministère de l’Enseignement Supérieur et de la Recherche Scientifique’’. We thank Dr. Souad Rouis and Mrs Dalel Ben farhat for their assistance during this study. References Abdelkefi-Mesrati, L., Tounsi, S., Jaoua, S., 2005. Characterization of a novel vip3type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol. Lett. 244, 353–358. Abdelkefi-Mesrati, L., Rouis, S., Sellami, S., Jaoua, S., 2009. Prays oleae midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3LB differs from that of Cry1Ac toxin. Mol. Biotechnol. 43, 15–19. Abdelkefi-Mesrati, L., Boukedi, H., Dammak-Karray, M., Sellami-Boudawara, T., Jaoua, S., Tounsi, S., 2011a. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. J. Invertebr. Pathol. 106, 250–254. Abdelkefi-Mesrati, L., Boukedi, H., Chakroun, M., Kamoun, F., Azzouz, H., Tounsi, S., Rouis, S., Jaoua, S., 2011b. Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin. J. Invertebr. Pathol. 107, 198–201. Banks, D.J., Jurat-Fuentes, J.L., Dean, D.H., Adang, M.J., 2001. Bacillus thuringiensis Cry1Ac and Cry1Fa d-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated. Insect Biochem. Mol. Biol. 31, 909–918.
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