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Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera
Accepted Manuscript Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera Hanen Boukedi, Saoussen Ben Khedher, Rania Hadhri, Samir Jaoua, Slim Tounsi, Lobna Abdelkefi-Mesrati PII:
S0041-0101(17)30064-8
DOI:
10.1016/j.toxicon.2017.02.018
Reference:
TOXCON 5574
To appear in:
Toxicon
Received Date: 9 December 2016 Revised Date:
15 February 2017
Accepted Date: 17 February 2017
Please cite this article as: Boukedi, H., Ben Khedher, S., Hadhri, R., Jaoua, S., Tounsi, S., AbdelkefiMesrati, L., Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera, Toxicon (2017), doi: 10.1016/j.toxicon.2017.02.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Vegetative insecticidal protein of Bacillus thuringiensis BLB459
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and its efficiency against Lepidoptera
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Hanen Boukedi1, Saoussen Ben Khedher1, Rania Hadhri1, Samir Jaoua2, Slim Tounsi1 and
Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box ‘‘2713’’, Doha, Qatar
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Laboratory of Biopesticides, Centre of Biotechnology of Sfax, Sfax, Tunisia
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Lobna Abdelkefi-Mesrati3,4*
Department of Biology, Faculty of Sciences and Arts-Khulais, University of Jeddah, Jeddah, Saudi Arabia
Higher Institute of Biotechnology of Sfax, Sfax, Tunisia
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*Author for correspondence: Dr. Lobna ABDELKEFI-MESRATI Tel. +966561137404 E-mail address: [email protected] 1
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ABSTRACT
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Bacillus thuringiensis strain BLB459 supernatant showed a promising activity against
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Lepidopteran pests with extremely damages in the larvae midgut. Investigations of the genes
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that encode secreted toxin demonstrated that this strain harbored a vip3-type gene named
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vip3(459). Based on its original nucleotide and amino acid sequences, this gene was cloned
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into pET-14b vector and overexpressed in Escherichia coli. The expressed protein was
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purified and tested against different insects and interestingly the novel toxin demonstrated a
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remarkable activity against the stored products pest Ephestia kuehniella and the polyphagous
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insects Spodoptera littoralis and Agrotis segetum. As demonstrated, the acute activity of
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Vip3(459) protein against A. segetum can be due to its original amino acids sequence and the
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putative receptors of this toxin in the larvae midgut. These results demonstrated that this Vip3
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toxin showed a wide spectrum of activity against Lepidoptera and support its use as a
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biological control agent.
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Keywords: Bacillus thuringiensis; Vegetative Insecticidal Protein; Lepidoptera; Biological
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control; Toxicity.
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ACCEPTED MANUSCRIPT 1. Introduction
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Bacillus thuringiensis is a Gram-positive bacterium which produces, during sporulation,
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crystalline inclusions containing one or more delta-endotoxins (Bravo et al., 2005). The latter
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are selectively toxic against a wide variety of insects (Bravo, 2007). These inclusions are
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solubilized in insect midgut, releasing proteins called δ-endotoxins that, upon proteolytic
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activation, exhibit a highly specific insecticidal activity (Höfte, 1989). In the past decades,
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many B. thuringiensis strains with different insects host spectra have been identified and their
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cry genes have been cloned in several microbes (Schnepf, 1998). Although industrial
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formulations of the Cry proteins have been used as biopesticides (Gelernter, 1990), most Cry
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proteins are not very effective in controlling some of agronomically important insects
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(Macintoch, 1990) such as the lepidopteran black cutworm (Agrotis ipsilon) attacking more
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than 50 crops including cereal grains (Rings, 1974). Extensive screening programs are being
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carried out by various groups to search for B. thuringiensis strains with new insecticidal
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spectra. These investigations have focused mainly on the identification of new insecticidal
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proteins that are expressed before and during sporulation. A second family of insecticidal
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proteins produced by B. thuringiensis during its vegetative growth phase (Vegetative
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insecticidal proteins: Vip) has been identified (Warren, 1997).
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The Vip proteins have no sequence homology with the Cry toxins and have been classified
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into three groups according to their sequence homology: Vip1, Vip2 and Vip3. Vip1 and Vip2
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proteins act as binary toxin and are toxic to Coleoptera (Shi et al., 2004) whereas Vip3
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proteins are active against Lepidoptera. Genes coding for these type of proteins have been
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found to be very common among B. thuringiensis isolates (Bhalla et al., 2005; Abdelkefi-
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Mesrati et al., 2005; Beard et al., 2008; Hernández-Rodríguez et al., 2009; Yu et al., 2011).
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The vip3A gene encodes an 88.5 kDa protein that is secreted into the supernatant fluid by B.
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thuringiensis cultures (Estruch, 1996). This protein possesses insecticidal activity against a
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ACCEPTED MANUSCRIPT wide spectrum of lepidopteran insects and displays acute bioactivity toward Agrotis ipsilon.
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Indeed, the activity of Vip3A protein against Agrotis ipsilon was 260 times higher than some
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of Cry1A proteins against this insect (Rings, 1974; Warren, 1997).
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As the environment is diverse, hence the insecticidal proteins are also diverse showing
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differential insecticidal activities. Therefore, it is necessary to screen more B. thuringiensis
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isolates to clone and characterize vip genes and their variants. The screening of 200 B.
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thuringiensis collection permitted the selection of a promising strain named BLB459 having
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an exceptional cry genes content (Boukedi et al., 2016a). In the present study, and to identify
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the origin of the potentiality of the secreted proteins of BLB459 strain in controlling
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lepidopteran pests, we decided to investigate the vip3 genes content and the effects of the
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corresponding protein on E. kuehniella, S. littoralis and A. segetum larvae.
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2. Materials and Methods
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2.1. Bacterial strains and growth conditions
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B. thuringiensis BLB459, known by its original cry genes content (Boukedi et al., 2016a), was
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used in the present study. For routine use in the laboratory, B. thuringiensis strains were
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grown in Luria-Bertani medium at 30 °C with shaking at 200 rpm.
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To promote protein production during the vegetative stage, cells were grown in PY Broth
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[1.2% (w/v) peptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 54 mM K2HPO4 and 16
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mM KH2PO4] (Donovan et al., 2001) at 30 °C then culture was centrifuged and the resulting
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supernatant was used for bioassays against Lepidopteran pests.
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Acrystalliferous isolates (Cry-, Spo+) were obtained by plasmid curing from the B.
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thuringiensis BLB459, by cultivating the wild strain at 42 °C for five days.
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2.2. Protein Quantification
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The concentrations of soluble proteins found in B. thuringiensis supernatants and purified
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Vip3(459) toxin were measured with the Bradford assay (Bio-Rad), using bovine serum
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albumin as a standard.
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2.3. Bioassays
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A free ingestion technique was used to assess the toxicity to E. kuehniella, S. littoralis and A.
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segetum larvae of B. thuringiensis supernatant extracts and purified Vip3(459) protein as
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described by Abdelkefi-Mesrati et al. (2011a,b) and Boukedi et al. (2015). The experiments
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were replicated three times with the presence of a negative control set maintained in the same
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conditions of temperature 23 °C, relative humidity of 65% and a photoperiod of 18 h light and
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(LC50) were calculated by probit analysis using programs written in the R. language
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(Venables and Smith, 2004).
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2.4. Preparation and sectioning of insect tissues
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After exposure to the B. thuringiensis supernatant extract, S. littoralis larvae were placed in
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formol solution (10%) then tissues were dehydrated using increasing ethanol concentrations
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and rinsed in 100% toluene solution. After paraffin wax embedding, five-micrometer sections
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were prepared and placed on slides coated with a mix of 1.5% egg albumin and 3% glycerol
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in distilled water. Then, tissues were de-paraffinated using 100% toluene solution and slides
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were stained with hematoxylineosin as described by Ruiz et al. (2004).
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2.5. DNA extraction and PCR amplification
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DNA was extracted from B. thuringiensis strains using the alkali lysis method including a
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lysozyme treatment step as described by Sambrook et al. (1989). Based on the published
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sequences of vip3-type genes, different oligonucleotides were designed (Table 1). In the PCR
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reaction, V1 and V2 primers (Table 1) were used for the detection of vip3-like genes using
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DNA extracted from B. thuringiensis strains as template and a ‘‘Gene Amp PCR System
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2700’’ (Applied Biosystems) (Jaoua et al., 1996).
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Using B. thuringiensis DNA, iQ SYBER Green Supermix (BIORAD) and (V13/PS21)
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primers (Table 1), Real-time quantitative PCR reactions were accomplished to amplify vip3-
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type genes. The amplification and detection of PCR products were performed with C1000
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thermal cycler (Bio-Rad) during 45 cycles (Wielinga et al., 2011).
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2.6. Cloning and sequencing of vip3-type gene
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ACCEPTED MANUSCRIPT The putative vip3-type gene of BLB459 was PCR amplified, using total DNA isolated from B.
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thuringiensis strain as template, primers V1 and V3 (Table 1) and DNA polymerase
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(Amersham). The amplified fragment, with a molecular weight of about 2.37 kb, was purified
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from the agarose gel. Then, the vip3 open reading frame (ORF) was cloned in pGEM-Teasy
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vector (Promega) generating a recombinant plasmid pGEMvip3(459). E. coli cells (Top10)
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transformation was performed as reported by Sambrook et al. (1989), then selection of E. coli
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transformants was performed on LB medium plates containing ampicillin (100 µg/ml), 5-
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bromo-4-chloro- 3-indolyl β-D-galactoside (40 µg/ml) and Isopropyl β-D-thiogalactoside (80
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µg/ml).
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The vip3(459) gene sequencing was carried out using the recombinant plasmid, the taq
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DyeDeoxy Terminator Cycle Sequencing kit and a 3700 ABI Prism DNA sequencer (Applied
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Biosystems, Foster City, CA) according to instructions of the manufacturer. Then, the
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obtained sequence was subjected to a blast nucleotide homology search against the nucleotide
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database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/
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Blast.cgi).
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2.7. Over-expression of vip3(459) in Escherichia coli and protein purification
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Overexpression of vip3(459) (GenBank Accession No. JN 990981) was achieved by cloning
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the ORF in the pET-14b vector (Novagen). Restriction enzyme sites NdeI and BamHI were
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created by PCR, respectively, upstream the initiation codon (ATG) and downstream the stop
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codon of the vip3 gene using primers VipM1 and VipM2 as described by Abdelkefi-Mesrati
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et al. (2009). After being cloned into pGEM-Teasy vector (Promega), the 2.37 kb fragment
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(ORF) was recovered by digesting it with NdeI and BamHI restriction enzymes then cloned in
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frame in its 5’ end with the His-tag sequence of the E. coli expression vector pET-14b
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(Novagen). The recombinant plasmid, named pET-vip3(459), was transformed into E. coli
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100 µg/ml ampicillin and induced using IPTG, as described by Abdelkefi-Mesrati et al.
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(2009).
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The cell pellet was resuspended in sonication buffer [PBS 19 (pH 7.5); 4 mM 4-(2-
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aminoethyl)-benzenesulfonyl fluoride] then sonicated (Abdelkefi-Mesrati et al., 2009) and
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centrifuged. To purify the Vip3(459) toxin fused with six histidine, the supernatant was
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loaded onto His-Trap column (Amersham) preequilibrated with a binding buffer (PBS 1X,
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imidazole 40 mM) then bound proteins were eluted using elution buffers containing
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increasing concentrations of imidazole in PBS 1X (Abdelkefi-Mesrati et al., 2009).
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2.8. A. segetum BBMV preparation and ligand-blotting
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Purified Vip3(459) proteins were activated by proteolysis using bovine pancreas trypsin
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(Amersham Pharmacia Biotech, France) as described by Abdelkefi-Mesrati et al. (2011b) then
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diluted in bicarbonate buffer (40 mM). After adding the biotinylation substrate (ECL™
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protein biotinylation module: Amersham Pharmacia Biotech, France), the mixture was
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incubated at room temperature for 1 h with agitation. Purification of the biotinylated toxin
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was operated by loading the mixture on G25 column and elution using PBS 1X, pH 7.5.
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Brush border membrane vesicles (BBMV) were prepared from A. segetum as described by
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Wolfersberger et al. (1987).
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For binding assays, 40 µg of BBMV were separated via SDS-PAGE then blotted by
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electrotransfer onto a nitrocellulose membrane (Bio-Rad, France). After being blocked with
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5% milk for 1 h, the membranes were reacted with biotinylated trypsinized toxins (40 nM) for
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2 h at room temperature then washed three times during 5 min as described by Abdelkefi-
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mesrati et al. (2011b). Membranes were incubated with streptavidin-peroxidase conjugate
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(1:1500 dilutions) for 1 h then binding was visualized using luminol according to
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2.9. Statistical analysis
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Data were expressed as means ± standard deviation (SD). The mortality was compared by
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using analysis of variances (ANOVA). The difference between individual means was deemed
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to be significant at P < 0.05. Statistical analyses were performed with SAS software (The SAS
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system for Windows ® 9.2, SAS Institute Inc., Cary, NC, USA).
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3. Results and Discussion
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3.1. Insecticidal activity of BLB459 supernatant
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When tested against first instar larvae of Lepidoptera, supernatant extract of BLB459 media
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culture showed a toxic action against E. kuehniella, S. littoralis and A. segetum with a
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percentage reaching the 80% (Table 2). This data demonstrated that B. thuringiensis strain
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BLB459 secreted, at the vegetative stage of its growth, molecules having insecticidal
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properties. According to Duncan test performed after ANOVA analysis, mortality caused by
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BLB459 supernatant on the tested lepidopteran pests was significantly higher than that of the
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reference strain B. thuringiensis BUPM95 (Abdelkefi-Mesrati et al., 2005) (Table 2). One of
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the suspected molecules was the vegetative insecticidal protein that can be secreted by some
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B. thuringiensis strains and particularly Vip3 toxins characterized by their activity against
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Lepidoptera (Estruch et al., 1996; Boukedi et al., 2016b).
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3.2. Histopathological effects of BLB459 supernatant on S. littoralis larvae midgut
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Histological observations of BLB459 supernatant effects on S. littoralis were studied on first
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instar larvae which had been fed a diet containing the supernatant extract. Compared to the
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uniform morphology and well-defined epithelial cells with unaffected apical microvilli
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membrane (Fig. 1A), extensive damages were detected in the midgut of larvae treated with
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BLB459 supernatant (Fig. 1B). Histopathological modifications included intensive
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vacuolization of the cytoplasm, brush border membrane destruction and vesicle secretion in
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the apical region of cells toward the midgut lumen (Fig. 1B). The deleterious histological
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observations detected in the midgut of the Lepidoptera S. littoralis demonstrated that this
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larvae is very sensitive to BLB459 supernatant. This kind of mode of action reminds us that
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described for B. thurigiensis Vip3 toxins active against Lepidoptera. In fact, Abdelkefi-
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Mesrati et al. (2011a,b) demonstrated that Vip3Aa16 of B. thuringiensis BUPM95 caused
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serious damages in the midgut larvae of S. littoralis and E. kuehniella.
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3.3. Evidence of the vip3 gene presence in BLB459 strain (PCR and Quantitative PCR)
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Based on results described above, we decided to search the presence of vip3-type gene in B.
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thuringiensis BLB459 genome using PCR investigation. Using V1 and V2 primers (Table 1)
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and in contrast to the negative control which did not lead to any amplification product,
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BLB459 DNA exhibited expected vip3 amplified fragment of about 0.4 kb demonstrating that
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this strain is Vip3+.
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To compare the copy numbers of vip3-type genes between B. thuringiensis BLB459 and the
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reference strain BUPM95 (Abdelkefi-Mesrati et al., 2005), we amplified identical quantities
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of DNA extracted from each strain by Real-time quantitative PCR. As shown in figure 2,
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fluorescence emerged early in the case of BLB459 DNA with a threshold cycle (Ct) of about
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24 (Fig. 2A) whereas the Ct detected for BUPM95 DNA was around 29 (Fig. 2B).
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Differences among the mean Ct values of the samples tested demonstrated that BLB459 strain
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contains a higher copy-number of vip3 gene than BUPM95 and such result may explain the
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Lepidoptera (Table 2).
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3.4. Localization of vip3 gene on the B. thuringiensis genome
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Plasmid curing was performed by cultivating the Vip3-producing B. thuringiensis strain
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BUPM459 at 42 °C for five days. Then, different clones were analyzed and acrystalliferous
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Cry- mutants were obtained with high frequency. DNA extracted from acrystalliferous clones
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demonstrated that B. thuringiensis BLB459 lost different plasmid after temperature treatment
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(Fig. 3A). Using PCR amplification and DNA extracted from different clones, we noted the
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absence of any amplification corresponding to cry genes. Interestingly, among all the tested
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clones Cry-, only one (Clone 3) showed an amplification using PS20 and PS21 primers (Fig.
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3B), demonstrating that this clone conserved its vip3 gene. These data evidenced that cry and
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vip3 genes are located on different plasmids in BLB459 genome and are lost at different
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moment during the curing treatment using high cultivating temperature. This result is original
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since Abdelkefi-Mesrati et al. (2005) demonstrated that cry and vip3 genes are located on a
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same megaplasmid of B. thuringiensis BUPM95.
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3.5. Cloning and sequence analysis of the BLB459 vip3 gene
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To know if the higher toxicity of BLB459 supernatant extract against Lepidoptera was due
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only to the greater copy number of its vip3 gene compared to that of BUPM95 or also to
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differences in the amino acids (AA) sequences of Vip3 proteins, the vip3 gene was cloned
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from this strain, characterized and compared with published vip3 genes. The amplified PCR
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fragment corresponding to the open reading frame (ORF) of vip3(459) was cloned in the
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pGEM-Teasy vector and the resultant recombinant plasmid was used for sequencing of vip3
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gene. The obtained sequence corresponds to an ORF of 2370 pb encoding a protein of about
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search for sequence similarity, we demonstrated that vip3(459) harbored some differences
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compared to the other reported vip3 genes. This result was confirmed with multiple
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alignments using CLUSTALW program. When compared with the known vip3-type genes,
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there was substitution at position 1106 of T for C (transition), resulting in the substitution of
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V(369) for A and a substitution at position 1786 of G for A (transition), resulting in the
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substitution of V(596) for I. Compared with vip3Aa16 of B. thuringiensis BLB459
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(Abdelkefi-Mesrati et al., 2005), there was another substitution at position 361 of C for A
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(transversion), resulting in the substitution of L(361) for I (Fig. 5).
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3.6. Over-expression of vip3(459) in Escherichia coli and protein purification
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The vip3(459) gene (GenBank Accession No. JN 990981) was cloned in the pET-14b vector
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(Novagen) as described above. Over-expression was performed using IPTG as inducer and E.
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coli (pET-vip3(459)) as recombinant strain. After sonication of IPTG-induced cells and non-
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induced E. coli, used as negative control, supernatant proteins were analyzed by SDS-PAGE.
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As shown on figure 4A, the two strains showed apparently similar protein patterns with a
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major exception of a 90-kDa Vip3-sized protein, which was produced only by the IPTG
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induced strain (Fig. 4A). The six-histidine tail fused at the N-terminal end of Vip3(459)
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protein allowed its purification using the His-trap column and applying increasing gradient of
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imidazole (Fig. 4B).
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3.7. Insecticidal efficiency of Vip3(459) on lepidopteran larvae
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When tested against first instar larvae of Lepidoptera, purified Vip3(459) protein showed
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toxicity with an LC50 of about 23, 30 and 288 ng/cm2, against E. kuehniella, A. segetum and S.
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littoralis, respectively (Table 3). Furthermore, non-killed larvae exposed to the purified
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negative control, did not affect larval growth increase, weight gain, or morphology. These
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data demonstrated that Vip3 toxin of BLB459 is active against these pests and can be used in
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a biological control program. When compared with Vip3Aa16 protein, the most studied Vip3
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toxin active against A. segetum with an LC50 of about 86 ng/cm2, we demonstrated that
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Vip3(459) protein was more efficient with an LC50 of about 30 ng/cm2 (Table 2). The
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important activity of Vip3(459) against A. segetum may be due to the 3 AA that differ
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between this the sequences of this toxin and Vip3Aa16 protein (Abdelkefi-Mesrati et al.,
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2005).
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3.8. Vip3(459) binding to A. segetum BBMV
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To know if the efficiency of Vip3(459) toxin was also influenced by another factor implicated
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in the Vip3 protein mode of action against Lepidoptera, we decided to investigate the proteins
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receptors recognized by this toxin in the larvae midgut of A. segetum by BBMV ligand
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blotting assays. The trypsin-activated form of Vip3(459) toxin produced by proteolysis of the
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purified protein (Fig. 4, Lane 5) was applied, after biotinylation, to study its interaction with
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BBMV. In contrast to the BSA blot, used as negative control, that showed no interaction with
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the biotinylated trypsinized Vip3(459) toxin (Fig. 6, Lane 2), the latter bound to 2 proteins of
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about 65 and 100 kDa (Fig. 6, Lane 1). These putative receptors (65 and 100 kDa) detected in
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the case of A. segetum differs from those recognized by the Vip3Aa16 toxin of B. turingiensis
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BUPM95 having a unique putative receptor of about 65 kDa in the BBMV of this Lepidoptera
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(Ben Hamadou-Charfi et al., 2013). This difference in the number of putative receptors
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recognized by these toxins could contribute to the variability in toxins efficiencies towards the
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same lepidopteran host.
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These results can explain the efficacy of the vegetative insecticidal protein produced by
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BLB459 strain against A. segetum and support the use of Vip3(459) as a biological control
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agent against a large spectrum of Lepidoptera.
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Acknowledgments
This work was supported by grants from the Tunisian Ministry of Higher Education and Scientific Research, Tunisia.
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protein Vip3Aa16 differs from that of Cry1Ac toxin. J. Invertebr. Pathol. 114. 139-143.
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2016a. Isolation and Characterization of a new Bacillus thuringiensis strain with a
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promising toxicity against Lepidopteran pests. Microbiol. Res. 186. 9-15.
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Ephestia kuehniella. J. Plant Dis. Prot. DOI 10.1007/s41348-016-0004-5.
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Bravo, A., Gill S.S., Soberón M., 2005. Bacillus thuringiensis Mechanisms and Use In: Comprehensive Molecular Insect Science. Elsevier BV. p. 175-206. Bravo, A., Gill S.S., Soberón M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt
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Hernández-Rodríguez, C.S., Boets A., Van Rie J., Ferré J., 2009. Screening and identification of vip genes in Bacillus thuringiensis strains. J. Appl. Microbiol. 107. 217-225. Höfte, H., Whiteley H.R., 1989. Insecticidal crystal proteins of Bacillus thuringiensis.
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Shi, Y., Xu W., Yuan M., Tang M., Chen J., Pang Y., 2004. Expression of vip1/vip2 genes in
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Appl. Microbiol. 97. 757-765.
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Venables, W.N., Smith D.M., 2004. The R. development core team. An introduction to R. version 1.9.1.
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Warren, G.W., 1997. Vegetative insecticidal proteins: novel proteins for control of corn pests.
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In advances in insect control, the role of transgenic plants ed. Carozzi, N.B. and
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Koziel, M. London: Taylors & Francis Ltd. p. 109-121.
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Wielinga, P.R., Hamidjaja, R.A., Ågren, J., Knutsson, R., Segerman, B., Fricker, M., Ehling-
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Schulz, M., de Groot, A., Burton, J., Brooks, T., Janse, I., van Rotterdam, B., 2011. A
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Int. J. Food Microbiol. doi:10.1016/j.ijfoodmicro.2010.07.039.
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Wolfersberger, M., Luthy P., Maurer A., Parenti P., Sacchi P.V., Giordana B., 1987.
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Preparation of brush border membrane vesicles (BBMV) from larval lepidopteran
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midgut. Comp. Biochem. Physiol. 86. 301-308.
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Yu, X., Zheng A., Zhu J., Wang S., Wang L., Deng Q., Li S., Liu H., Li P., 2011.
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Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis
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from Shichuan Basin in China. Curr. Microbiol. 62. 752-757.
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Fig. 1. Histopathological effects of BLB459 supernatant on Lepidopteran midgut larvae.
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A: General aspects of the midgut larvae of S. littoralis; B: Histopathological effects of
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supernatant extract on the midgut larvae of S. littoralis. Lu, lumen; Am, Apical membrane;
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Bm, Basement membrane; V, vacuole formation. Magnification 40×. Arrowhead indicates
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vesicle formation.
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Fig. 2. The amplification curves of vip3 gene as recorded in Real-time quantitative PCR
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reactions. Reactions operated using DNA extracted from B. thuringiensis strains BUPM95
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(A) and BLB459 (B).
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RFU: relative fluorescence units.
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Fig. 3. vip3 gene localization in the B. thuringiensis BLB459 genome. (A) Plasmids
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contents of BLB459 strain and its mutants Cry-. Lanes: 1, B. thuringiensis BLB459; 2, 3 and
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4, three different clones (1, 2 and 3) obtained by cultivating the wild strain at 42 °C for five
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days. (B) PCR amplification using PS20 and PS21 primers. Lanes: 1, DNA molecular weight
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marker (λ−PstI); 2, 1456 pb product obtained using DNA extracted from clone 3; 3, negative
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control.
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P indicates the position of the plasmid carrying vip3 gene of B. thuringiensis BLB459.
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Fig. 4. Heterologous expression of vip3(459) in E. coli and protein purification. (A) SDS-
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PAGE analysis of E. coli supernatants. Lanes: 1, Molecular weight markers; 2, Supernatant
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proteins of sonicated non-induced recombinant E. coli cells; 3, Supernatant proteins of
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sonicated IPTG-induced recombinant E. coli cells. (B) SDS-PAGE stained with Coomassie
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stain. Lanes: 1, Supernatant proteins of sonicated IPTG-induced E. coli cells containing pET-
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increasing concentration of imidazole: 100, 200 and 300 mM, respectively.
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Fig. 5. Comparison of the Vip3(459) amino acid sequence with those encoded by the
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other vip3-type genes.
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GenBank Accession Numbers of the vip3 genes and their corresponding proteins: 1, Vip3V
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(AAN60738.1); 2, Vip184 (AAO32350.1);
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(CAI96522.1); 5 Vip3 (CAA76665.1); 6, Vip3 (AAU89707.1); 7, Vip3Aa16 (AAW65132)
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Vertical downward arrows indicate amino acids positions, the black boxes represent the
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residues showing variations and only regions containing differences are presented.
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Fig. 6. A. segetum putative receptors of Vip3(459) toxins. Lanes: 1, A. segetum BBMV
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ligand blotting with biotinylated Vip3(459) toxins; 2, Bovine serum albumin (BSA) used as
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negative control.
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3, Vip3BR (AAW62286.1); 4, Vip3
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ACCEPTED MANUSCRIPT Table 1. Primers used in PCR.
Sequence (5’ 3’) ATGAACAAGAATAATACTA TCTATTTGCAGACTTAGCGC TTACTTAATAGAGACATCGT AAGATGCA*TATGAACAAGAATAATA GATG*GAT CCCGATCTTACTTAATAG CAAGCCGCAAATCTTGTGGA CATTCCACGATGTAATGGTAGG ATGGCTTGTTTCGCTACATC
Abdelkefi-Mesrati et al. (2005)
Abdelkefi-Mesrati et al. (2009) CAA68485 L48811
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V1 (f) V2 (b) V3 (b) VipM1 (f) VipM2 (b) V13 (f) PS20 (f) PS21 (b)
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(b) backward primers
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ACCEPTED MANUSCRIPT Table 2: Insecticidal activities of B. thuringiensis supernatants
Strains Lepidoptera BLB459
E. kuhenilla
75.30 ± 0.40% b
85.40 ± 0.41% a
S. littoralis
76.50 ± 0.43% b
82.50 ± 0.51% a
A. segetum
72.50 ± 0.10% b
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BUPM95
79.16 ± 0.54% a
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* Means in the same column not followed by the same letters are significantly different at p =
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0.05 upon Duncan multiple range test.
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ACCEPTED MANUSCRIPT Table 3: Insecticidal efficiency of Vip3(459) on lepidopteran larvae
Toxins 2
CL50 (ng/cm ) Vip3-459
Vip3Aa16
E. kuehniella
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36 (+/- 25) (Abdelkefi-Mesrati et al., 2011 a) 305 (+/-95) S. littoralis
288 (+/- 31)
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(Abdelkefi-Mesrati et al., 2011 b) 86 (+/- 10)
A. segetum
30 (+/- 5)
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- Bacillus thuringiensis strain BLB459 supernatant is active against Lepidoptera
- B. thuringiensis strain BLB459 harbored a vip3-type gene named vip3(459) - Vip3(459) toxin demonstrated a remarkable activity against lepidopteran pests
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- The acute activity of Vip3(459) against A. segetum can be due to its sequence
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- This activity can be also due to the putative receptors of this toxin in the midgut
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I testify on behalf of all co-authors that our article submitted to Toxicon Title: Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera
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All authors: Hanen Boukedi, Rania Hadhri, Samir Jaoua, Slim Tounsi and Lobna Abdelkefi-Mesrati
Date: 12 December 2016
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The Corresponding author: Dr. Lobna Abdelkefi-Mesrati
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1) this material has not been published in whole or in part elsewhere; 2) the manuscript is not currently being considered for publication in another journal; 3) all authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content.
S0041-0101(17)30064-8
DOI:
10.1016/j.toxicon.2017.02.018
Reference:
TOXCON 5574
To appear in:
Toxicon
Received Date: 9 December 2016 Revised Date:
15 February 2017
Accepted Date: 17 February 2017
Please cite this article as: Boukedi, H., Ben Khedher, S., Hadhri, R., Jaoua, S., Tounsi, S., AbdelkefiMesrati, L., Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera, Toxicon (2017), doi: 10.1016/j.toxicon.2017.02.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Vegetative insecticidal protein of Bacillus thuringiensis BLB459
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and its efficiency against Lepidoptera
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Hanen Boukedi1, Saoussen Ben Khedher1, Rania Hadhri1, Samir Jaoua2, Slim Tounsi1 and
Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box ‘‘2713’’, Doha, Qatar
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Laboratory of Biopesticides, Centre of Biotechnology of Sfax, Sfax, Tunisia
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Lobna Abdelkefi-Mesrati3,4*
Department of Biology, Faculty of Sciences and Arts-Khulais, University of Jeddah, Jeddah, Saudi Arabia
Higher Institute of Biotechnology of Sfax, Sfax, Tunisia
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*Author for correspondence: Dr. Lobna ABDELKEFI-MESRATI Tel. +966561137404 E-mail address: [email protected] 1
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ABSTRACT
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Bacillus thuringiensis strain BLB459 supernatant showed a promising activity against
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Lepidopteran pests with extremely damages in the larvae midgut. Investigations of the genes
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that encode secreted toxin demonstrated that this strain harbored a vip3-type gene named
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vip3(459). Based on its original nucleotide and amino acid sequences, this gene was cloned
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into pET-14b vector and overexpressed in Escherichia coli. The expressed protein was
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purified and tested against different insects and interestingly the novel toxin demonstrated a
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remarkable activity against the stored products pest Ephestia kuehniella and the polyphagous
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insects Spodoptera littoralis and Agrotis segetum. As demonstrated, the acute activity of
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Vip3(459) protein against A. segetum can be due to its original amino acids sequence and the
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putative receptors of this toxin in the larvae midgut. These results demonstrated that this Vip3
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toxin showed a wide spectrum of activity against Lepidoptera and support its use as a
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biological control agent.
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Keywords: Bacillus thuringiensis; Vegetative Insecticidal Protein; Lepidoptera; Biological
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control; Toxicity.
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Bacillus thuringiensis is a Gram-positive bacterium which produces, during sporulation,
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crystalline inclusions containing one or more delta-endotoxins (Bravo et al., 2005). The latter
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are selectively toxic against a wide variety of insects (Bravo, 2007). These inclusions are
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solubilized in insect midgut, releasing proteins called δ-endotoxins that, upon proteolytic
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activation, exhibit a highly specific insecticidal activity (Höfte, 1989). In the past decades,
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many B. thuringiensis strains with different insects host spectra have been identified and their
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cry genes have been cloned in several microbes (Schnepf, 1998). Although industrial
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formulations of the Cry proteins have been used as biopesticides (Gelernter, 1990), most Cry
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proteins are not very effective in controlling some of agronomically important insects
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(Macintoch, 1990) such as the lepidopteran black cutworm (Agrotis ipsilon) attacking more
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than 50 crops including cereal grains (Rings, 1974). Extensive screening programs are being
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carried out by various groups to search for B. thuringiensis strains with new insecticidal
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spectra. These investigations have focused mainly on the identification of new insecticidal
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proteins that are expressed before and during sporulation. A second family of insecticidal
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proteins produced by B. thuringiensis during its vegetative growth phase (Vegetative
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insecticidal proteins: Vip) has been identified (Warren, 1997).
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The Vip proteins have no sequence homology with the Cry toxins and have been classified
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into three groups according to their sequence homology: Vip1, Vip2 and Vip3. Vip1 and Vip2
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proteins act as binary toxin and are toxic to Coleoptera (Shi et al., 2004) whereas Vip3
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proteins are active against Lepidoptera. Genes coding for these type of proteins have been
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found to be very common among B. thuringiensis isolates (Bhalla et al., 2005; Abdelkefi-
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Mesrati et al., 2005; Beard et al., 2008; Hernández-Rodríguez et al., 2009; Yu et al., 2011).
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The vip3A gene encodes an 88.5 kDa protein that is secreted into the supernatant fluid by B.
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thuringiensis cultures (Estruch, 1996). This protein possesses insecticidal activity against a
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Indeed, the activity of Vip3A protein against Agrotis ipsilon was 260 times higher than some
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of Cry1A proteins against this insect (Rings, 1974; Warren, 1997).
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As the environment is diverse, hence the insecticidal proteins are also diverse showing
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differential insecticidal activities. Therefore, it is necessary to screen more B. thuringiensis
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isolates to clone and characterize vip genes and their variants. The screening of 200 B.
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thuringiensis collection permitted the selection of a promising strain named BLB459 having
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an exceptional cry genes content (Boukedi et al., 2016a). In the present study, and to identify
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the origin of the potentiality of the secreted proteins of BLB459 strain in controlling
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lepidopteran pests, we decided to investigate the vip3 genes content and the effects of the
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corresponding protein on E. kuehniella, S. littoralis and A. segetum larvae.
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2. Materials and Methods
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2.1. Bacterial strains and growth conditions
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B. thuringiensis BLB459, known by its original cry genes content (Boukedi et al., 2016a), was
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used in the present study. For routine use in the laboratory, B. thuringiensis strains were
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grown in Luria-Bertani medium at 30 °C with shaking at 200 rpm.
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To promote protein production during the vegetative stage, cells were grown in PY Broth
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[1.2% (w/v) peptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 54 mM K2HPO4 and 16
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mM KH2PO4] (Donovan et al., 2001) at 30 °C then culture was centrifuged and the resulting
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supernatant was used for bioassays against Lepidopteran pests.
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Acrystalliferous isolates (Cry-, Spo+) were obtained by plasmid curing from the B.
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thuringiensis BLB459, by cultivating the wild strain at 42 °C for five days.
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2.2. Protein Quantification
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The concentrations of soluble proteins found in B. thuringiensis supernatants and purified
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Vip3(459) toxin were measured with the Bradford assay (Bio-Rad), using bovine serum
69
albumin as a standard.
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2.3. Bioassays
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A free ingestion technique was used to assess the toxicity to E. kuehniella, S. littoralis and A.
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segetum larvae of B. thuringiensis supernatant extracts and purified Vip3(459) protein as
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described by Abdelkefi-Mesrati et al. (2011a,b) and Boukedi et al. (2015). The experiments
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were replicated three times with the presence of a negative control set maintained in the same
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conditions of temperature 23 °C, relative humidity of 65% and a photoperiod of 18 h light and
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(LC50) were calculated by probit analysis using programs written in the R. language
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(Venables and Smith, 2004).
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2.4. Preparation and sectioning of insect tissues
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After exposure to the B. thuringiensis supernatant extract, S. littoralis larvae were placed in
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formol solution (10%) then tissues were dehydrated using increasing ethanol concentrations
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and rinsed in 100% toluene solution. After paraffin wax embedding, five-micrometer sections
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were prepared and placed on slides coated with a mix of 1.5% egg albumin and 3% glycerol
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in distilled water. Then, tissues were de-paraffinated using 100% toluene solution and slides
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were stained with hematoxylineosin as described by Ruiz et al. (2004).
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2.5. DNA extraction and PCR amplification
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DNA was extracted from B. thuringiensis strains using the alkali lysis method including a
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lysozyme treatment step as described by Sambrook et al. (1989). Based on the published
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sequences of vip3-type genes, different oligonucleotides were designed (Table 1). In the PCR
90
reaction, V1 and V2 primers (Table 1) were used for the detection of vip3-like genes using
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DNA extracted from B. thuringiensis strains as template and a ‘‘Gene Amp PCR System
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2700’’ (Applied Biosystems) (Jaoua et al., 1996).
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Using B. thuringiensis DNA, iQ SYBER Green Supermix (BIORAD) and (V13/PS21)
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primers (Table 1), Real-time quantitative PCR reactions were accomplished to amplify vip3-
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type genes. The amplification and detection of PCR products were performed with C1000
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thermal cycler (Bio-Rad) during 45 cycles (Wielinga et al., 2011).
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2.6. Cloning and sequencing of vip3-type gene
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thuringiensis strain as template, primers V1 and V3 (Table 1) and DNA polymerase
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(Amersham). The amplified fragment, with a molecular weight of about 2.37 kb, was purified
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from the agarose gel. Then, the vip3 open reading frame (ORF) was cloned in pGEM-Teasy
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vector (Promega) generating a recombinant plasmid pGEMvip3(459). E. coli cells (Top10)
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transformation was performed as reported by Sambrook et al. (1989), then selection of E. coli
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transformants was performed on LB medium plates containing ampicillin (100 µg/ml), 5-
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bromo-4-chloro- 3-indolyl β-D-galactoside (40 µg/ml) and Isopropyl β-D-thiogalactoside (80
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µg/ml).
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The vip3(459) gene sequencing was carried out using the recombinant plasmid, the taq
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DyeDeoxy Terminator Cycle Sequencing kit and a 3700 ABI Prism DNA sequencer (Applied
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Biosystems, Foster City, CA) according to instructions of the manufacturer. Then, the
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obtained sequence was subjected to a blast nucleotide homology search against the nucleotide
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database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/
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Blast.cgi).
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2.7. Over-expression of vip3(459) in Escherichia coli and protein purification
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Overexpression of vip3(459) (GenBank Accession No. JN 990981) was achieved by cloning
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the ORF in the pET-14b vector (Novagen). Restriction enzyme sites NdeI and BamHI were
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created by PCR, respectively, upstream the initiation codon (ATG) and downstream the stop
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codon of the vip3 gene using primers VipM1 and VipM2 as described by Abdelkefi-Mesrati
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et al. (2009). After being cloned into pGEM-Teasy vector (Promega), the 2.37 kb fragment
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(ORF) was recovered by digesting it with NdeI and BamHI restriction enzymes then cloned in
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frame in its 5’ end with the His-tag sequence of the E. coli expression vector pET-14b
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(Novagen). The recombinant plasmid, named pET-vip3(459), was transformed into E. coli
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100 µg/ml ampicillin and induced using IPTG, as described by Abdelkefi-Mesrati et al.
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(2009).
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The cell pellet was resuspended in sonication buffer [PBS 19 (pH 7.5); 4 mM 4-(2-
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aminoethyl)-benzenesulfonyl fluoride] then sonicated (Abdelkefi-Mesrati et al., 2009) and
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centrifuged. To purify the Vip3(459) toxin fused with six histidine, the supernatant was
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loaded onto His-Trap column (Amersham) preequilibrated with a binding buffer (PBS 1X,
129
imidazole 40 mM) then bound proteins were eluted using elution buffers containing
130
increasing concentrations of imidazole in PBS 1X (Abdelkefi-Mesrati et al., 2009).
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2.8. A. segetum BBMV preparation and ligand-blotting
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Purified Vip3(459) proteins were activated by proteolysis using bovine pancreas trypsin
133
(Amersham Pharmacia Biotech, France) as described by Abdelkefi-Mesrati et al. (2011b) then
134
diluted in bicarbonate buffer (40 mM). After adding the biotinylation substrate (ECL™
135
protein biotinylation module: Amersham Pharmacia Biotech, France), the mixture was
136
incubated at room temperature for 1 h with agitation. Purification of the biotinylated toxin
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was operated by loading the mixture on G25 column and elution using PBS 1X, pH 7.5.
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Brush border membrane vesicles (BBMV) were prepared from A. segetum as described by
139
Wolfersberger et al. (1987).
140
For binding assays, 40 µg of BBMV were separated via SDS-PAGE then blotted by
141
electrotransfer onto a nitrocellulose membrane (Bio-Rad, France). After being blocked with
142
5% milk for 1 h, the membranes were reacted with biotinylated trypsinized toxins (40 nM) for
143
2 h at room temperature then washed three times during 5 min as described by Abdelkefi-
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mesrati et al. (2011b). Membranes were incubated with streptavidin-peroxidase conjugate
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(1:1500 dilutions) for 1 h then binding was visualized using luminol according to
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2.9. Statistical analysis
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Data were expressed as means ± standard deviation (SD). The mortality was compared by
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using analysis of variances (ANOVA). The difference between individual means was deemed
150
to be significant at P < 0.05. Statistical analyses were performed with SAS software (The SAS
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system for Windows ® 9.2, SAS Institute Inc., Cary, NC, USA).
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3. Results and Discussion
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3.1. Insecticidal activity of BLB459 supernatant
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When tested against first instar larvae of Lepidoptera, supernatant extract of BLB459 media
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culture showed a toxic action against E. kuehniella, S. littoralis and A. segetum with a
156
percentage reaching the 80% (Table 2). This data demonstrated that B. thuringiensis strain
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BLB459 secreted, at the vegetative stage of its growth, molecules having insecticidal
158
properties. According to Duncan test performed after ANOVA analysis, mortality caused by
159
BLB459 supernatant on the tested lepidopteran pests was significantly higher than that of the
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reference strain B. thuringiensis BUPM95 (Abdelkefi-Mesrati et al., 2005) (Table 2). One of
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the suspected molecules was the vegetative insecticidal protein that can be secreted by some
162
B. thuringiensis strains and particularly Vip3 toxins characterized by their activity against
163
Lepidoptera (Estruch et al., 1996; Boukedi et al., 2016b).
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3.2. Histopathological effects of BLB459 supernatant on S. littoralis larvae midgut
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Histological observations of BLB459 supernatant effects on S. littoralis were studied on first
166
instar larvae which had been fed a diet containing the supernatant extract. Compared to the
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uniform morphology and well-defined epithelial cells with unaffected apical microvilli
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membrane (Fig. 1A), extensive damages were detected in the midgut of larvae treated with
170
BLB459 supernatant (Fig. 1B). Histopathological modifications included intensive
171
vacuolization of the cytoplasm, brush border membrane destruction and vesicle secretion in
172
the apical region of cells toward the midgut lumen (Fig. 1B). The deleterious histological
173
observations detected in the midgut of the Lepidoptera S. littoralis demonstrated that this
174
larvae is very sensitive to BLB459 supernatant. This kind of mode of action reminds us that
175
described for B. thurigiensis Vip3 toxins active against Lepidoptera. In fact, Abdelkefi-
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Mesrati et al. (2011a,b) demonstrated that Vip3Aa16 of B. thuringiensis BUPM95 caused
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serious damages in the midgut larvae of S. littoralis and E. kuehniella.
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3.3. Evidence of the vip3 gene presence in BLB459 strain (PCR and Quantitative PCR)
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Based on results described above, we decided to search the presence of vip3-type gene in B.
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thuringiensis BLB459 genome using PCR investigation. Using V1 and V2 primers (Table 1)
181
and in contrast to the negative control which did not lead to any amplification product,
182
BLB459 DNA exhibited expected vip3 amplified fragment of about 0.4 kb demonstrating that
183
this strain is Vip3+.
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To compare the copy numbers of vip3-type genes between B. thuringiensis BLB459 and the
185
reference strain BUPM95 (Abdelkefi-Mesrati et al., 2005), we amplified identical quantities
186
of DNA extracted from each strain by Real-time quantitative PCR. As shown in figure 2,
187
fluorescence emerged early in the case of BLB459 DNA with a threshold cycle (Ct) of about
188
24 (Fig. 2A) whereas the Ct detected for BUPM95 DNA was around 29 (Fig. 2B).
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Differences among the mean Ct values of the samples tested demonstrated that BLB459 strain
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contains a higher copy-number of vip3 gene than BUPM95 and such result may explain the
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Lepidoptera (Table 2).
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3.4. Localization of vip3 gene on the B. thuringiensis genome
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Plasmid curing was performed by cultivating the Vip3-producing B. thuringiensis strain
195
BUPM459 at 42 °C for five days. Then, different clones were analyzed and acrystalliferous
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Cry- mutants were obtained with high frequency. DNA extracted from acrystalliferous clones
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demonstrated that B. thuringiensis BLB459 lost different plasmid after temperature treatment
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(Fig. 3A). Using PCR amplification and DNA extracted from different clones, we noted the
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absence of any amplification corresponding to cry genes. Interestingly, among all the tested
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clones Cry-, only one (Clone 3) showed an amplification using PS20 and PS21 primers (Fig.
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3B), demonstrating that this clone conserved its vip3 gene. These data evidenced that cry and
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vip3 genes are located on different plasmids in BLB459 genome and are lost at different
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moment during the curing treatment using high cultivating temperature. This result is original
204
since Abdelkefi-Mesrati et al. (2005) demonstrated that cry and vip3 genes are located on a
205
same megaplasmid of B. thuringiensis BUPM95.
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3.5. Cloning and sequence analysis of the BLB459 vip3 gene
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To know if the higher toxicity of BLB459 supernatant extract against Lepidoptera was due
208
only to the greater copy number of its vip3 gene compared to that of BUPM95 or also to
209
differences in the amino acids (AA) sequences of Vip3 proteins, the vip3 gene was cloned
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from this strain, characterized and compared with published vip3 genes. The amplified PCR
211
fragment corresponding to the open reading frame (ORF) of vip3(459) was cloned in the
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pGEM-Teasy vector and the resultant recombinant plasmid was used for sequencing of vip3
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gene. The obtained sequence corresponds to an ORF of 2370 pb encoding a protein of about
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search for sequence similarity, we demonstrated that vip3(459) harbored some differences
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compared to the other reported vip3 genes. This result was confirmed with multiple
217
alignments using CLUSTALW program. When compared with the known vip3-type genes,
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there was substitution at position 1106 of T for C (transition), resulting in the substitution of
219
V(369) for A and a substitution at position 1786 of G for A (transition), resulting in the
220
substitution of V(596) for I. Compared with vip3Aa16 of B. thuringiensis BLB459
221
(Abdelkefi-Mesrati et al., 2005), there was another substitution at position 361 of C for A
222
(transversion), resulting in the substitution of L(361) for I (Fig. 5).
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3.6. Over-expression of vip3(459) in Escherichia coli and protein purification
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The vip3(459) gene (GenBank Accession No. JN 990981) was cloned in the pET-14b vector
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(Novagen) as described above. Over-expression was performed using IPTG as inducer and E.
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coli (pET-vip3(459)) as recombinant strain. After sonication of IPTG-induced cells and non-
227
induced E. coli, used as negative control, supernatant proteins were analyzed by SDS-PAGE.
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As shown on figure 4A, the two strains showed apparently similar protein patterns with a
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major exception of a 90-kDa Vip3-sized protein, which was produced only by the IPTG
230
induced strain (Fig. 4A). The six-histidine tail fused at the N-terminal end of Vip3(459)
231
protein allowed its purification using the His-trap column and applying increasing gradient of
232
imidazole (Fig. 4B).
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3.7. Insecticidal efficiency of Vip3(459) on lepidopteran larvae
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When tested against first instar larvae of Lepidoptera, purified Vip3(459) protein showed
235
toxicity with an LC50 of about 23, 30 and 288 ng/cm2, against E. kuehniella, A. segetum and S.
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littoralis, respectively (Table 3). Furthermore, non-killed larvae exposed to the purified
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negative control, did not affect larval growth increase, weight gain, or morphology. These
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data demonstrated that Vip3 toxin of BLB459 is active against these pests and can be used in
240
a biological control program. When compared with Vip3Aa16 protein, the most studied Vip3
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toxin active against A. segetum with an LC50 of about 86 ng/cm2, we demonstrated that
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Vip3(459) protein was more efficient with an LC50 of about 30 ng/cm2 (Table 2). The
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important activity of Vip3(459) against A. segetum may be due to the 3 AA that differ
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between this the sequences of this toxin and Vip3Aa16 protein (Abdelkefi-Mesrati et al.,
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2005).
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3.8. Vip3(459) binding to A. segetum BBMV
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To know if the efficiency of Vip3(459) toxin was also influenced by another factor implicated
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in the Vip3 protein mode of action against Lepidoptera, we decided to investigate the proteins
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receptors recognized by this toxin in the larvae midgut of A. segetum by BBMV ligand
250
blotting assays. The trypsin-activated form of Vip3(459) toxin produced by proteolysis of the
251
purified protein (Fig. 4, Lane 5) was applied, after biotinylation, to study its interaction with
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BBMV. In contrast to the BSA blot, used as negative control, that showed no interaction with
253
the biotinylated trypsinized Vip3(459) toxin (Fig. 6, Lane 2), the latter bound to 2 proteins of
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about 65 and 100 kDa (Fig. 6, Lane 1). These putative receptors (65 and 100 kDa) detected in
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the case of A. segetum differs from those recognized by the Vip3Aa16 toxin of B. turingiensis
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BUPM95 having a unique putative receptor of about 65 kDa in the BBMV of this Lepidoptera
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(Ben Hamadou-Charfi et al., 2013). This difference in the number of putative receptors
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recognized by these toxins could contribute to the variability in toxins efficiencies towards the
259
same lepidopteran host.
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These results can explain the efficacy of the vegetative insecticidal protein produced by
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BLB459 strain against A. segetum and support the use of Vip3(459) as a biological control
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agent against a large spectrum of Lepidoptera.
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Acknowledgments
This work was supported by grants from the Tunisian Ministry of Higher Education and Scientific Research, Tunisia.
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Fig. 1. Histopathological effects of BLB459 supernatant on Lepidopteran midgut larvae.
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A: General aspects of the midgut larvae of S. littoralis; B: Histopathological effects of
359
supernatant extract on the midgut larvae of S. littoralis. Lu, lumen; Am, Apical membrane;
360
Bm, Basement membrane; V, vacuole formation. Magnification 40×. Arrowhead indicates
361
vesicle formation.
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Fig. 2. The amplification curves of vip3 gene as recorded in Real-time quantitative PCR
363
reactions. Reactions operated using DNA extracted from B. thuringiensis strains BUPM95
364
(A) and BLB459 (B).
365
RFU: relative fluorescence units.
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Fig. 3. vip3 gene localization in the B. thuringiensis BLB459 genome. (A) Plasmids
367
contents of BLB459 strain and its mutants Cry-. Lanes: 1, B. thuringiensis BLB459; 2, 3 and
368
4, three different clones (1, 2 and 3) obtained by cultivating the wild strain at 42 °C for five
369
days. (B) PCR amplification using PS20 and PS21 primers. Lanes: 1, DNA molecular weight
370
marker (λ−PstI); 2, 1456 pb product obtained using DNA extracted from clone 3; 3, negative
371
control.
372
P indicates the position of the plasmid carrying vip3 gene of B. thuringiensis BLB459.
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Fig. 4. Heterologous expression of vip3(459) in E. coli and protein purification. (A) SDS-
374
PAGE analysis of E. coli supernatants. Lanes: 1, Molecular weight markers; 2, Supernatant
375
proteins of sonicated non-induced recombinant E. coli cells; 3, Supernatant proteins of
376
sonicated IPTG-induced recombinant E. coli cells. (B) SDS-PAGE stained with Coomassie
377
stain. Lanes: 1, Supernatant proteins of sonicated IPTG-induced E. coli cells containing pET-
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379
increasing concentration of imidazole: 100, 200 and 300 mM, respectively.
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Fig. 5. Comparison of the Vip3(459) amino acid sequence with those encoded by the
381
other vip3-type genes.
382
GenBank Accession Numbers of the vip3 genes and their corresponding proteins: 1, Vip3V
383
(AAN60738.1); 2, Vip184 (AAO32350.1);
384
(CAI96522.1); 5 Vip3 (CAA76665.1); 6, Vip3 (AAU89707.1); 7, Vip3Aa16 (AAW65132)
385
Vertical downward arrows indicate amino acids positions, the black boxes represent the
386
residues showing variations and only regions containing differences are presented.
387
Fig. 6. A. segetum putative receptors of Vip3(459) toxins. Lanes: 1, A. segetum BBMV
388
ligand blotting with biotinylated Vip3(459) toxins; 2, Bovine serum albumin (BSA) used as
389
negative control.
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3, Vip3BR (AAW62286.1); 4, Vip3
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ACCEPTED MANUSCRIPT Table 1. Primers used in PCR.
Sequence (5’ 3’) ATGAACAAGAATAATACTA TCTATTTGCAGACTTAGCGC TTACTTAATAGAGACATCGT AAGATGCA*TATGAACAAGAATAATA GATG*GAT CCCGATCTTACTTAATAG CAAGCCGCAAATCTTGTGGA CATTCCACGATGTAATGGTAGG ATGGCTTGTTTCGCTACATC
Abdelkefi-Mesrati et al. (2005)
Abdelkefi-Mesrati et al. (2009) CAA68485 L48811
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V1 (f) V2 (b) V3 (b) VipM1 (f) VipM2 (b) V13 (f) PS20 (f) PS21 (b)
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ACCEPTED MANUSCRIPT Table 2: Insecticidal activities of B. thuringiensis supernatants
Strains Lepidoptera BLB459
E. kuhenilla
75.30 ± 0.40% b
85.40 ± 0.41% a
S. littoralis
76.50 ± 0.43% b
82.50 ± 0.51% a
A. segetum
72.50 ± 0.10% b
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79.16 ± 0.54% a
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* Means in the same column not followed by the same letters are significantly different at p =
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0.05 upon Duncan multiple range test.
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ACCEPTED MANUSCRIPT Table 3: Insecticidal efficiency of Vip3(459) on lepidopteran larvae
Toxins 2
CL50 (ng/cm ) Vip3-459
Vip3Aa16
E. kuehniella
23 (+/- 4)
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288 (+/- 31)
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(Abdelkefi-Mesrati et al., 2011 b) 86 (+/- 10)
A. segetum
30 (+/- 5)
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- Bacillus thuringiensis strain BLB459 supernatant is active against Lepidoptera
- B. thuringiensis strain BLB459 harbored a vip3-type gene named vip3(459) - Vip3(459) toxin demonstrated a remarkable activity against lepidopteran pests
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- The acute activity of Vip3(459) against A. segetum can be due to its sequence
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- This activity can be also due to the putative receptors of this toxin in the midgut
ACCEPTED MANUSCRIPT
I testify on behalf of all co-authors that our article submitted to Toxicon Title: Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera
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All authors: Hanen Boukedi, Rania Hadhri, Samir Jaoua, Slim Tounsi and Lobna Abdelkefi-Mesrati
Date: 12 December 2016
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The Corresponding author: Dr. Lobna Abdelkefi-Mesrati
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1) this material has not been published in whole or in part elsewhere; 2) the manuscript is not currently being considered for publication in another journal; 3) all authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content.