RESEARCH Prays oleae Midgut Putative Receptor of Bacillus thuringiensis Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin Lobna Abdelkefi-Mesrati Æ Souad Rouis Æ Sameh Sellami Æ Samir Jaoua Published online: 12 May 2009 Ó Humana Press 2009 Abstract Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thurin- giensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expres- sed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particu- larly in case of resistance to Cry toxins. Keywords Bacillus thuringiensis Á Vegetative insecticidal protein Á Overexpression Á Midgut putative receptor Á Biological control Abbreviations B Bacillus P Prays IgG Immunoglobulin G Introduction The ability of Bacillus thuringiensis to produce parasporal crystals composed of insecticidal d-endotoxins has been the subject of numerous studies [1–3]. On the other hand, a number of secreted insecticidal proteins called vegetative insecticidal proteins (Vip) have been identified during the vegetative growth phase of B. thuringiensis strains [4–7]. This class of insecticidal proteins includes the toxins Vip1- Vip2 active on Coleoptera and Vip3 active on Lepidoptera [4, 5, 7–9]. Bacillus thuringiensis d-endotoxins have been suc- cessfully used to control many crop pests by either traditional spray applications or transgenic plant approa- ches. However, a number of cases of insect resistance to the B. thuringiensis d-endotoxins have been reported as a result of laboratory and, more rarely, field selections [10]. Binding of B. thuringiensis toxins to the receptors located on the epithelial brush border membrane has been identified as a critical step in their action and also the modification of this binding has been identified as a likely resistance mechanism [10, 11]. Therefore, searching for a new family of insecticidal toxins, with a mode of action or midgut receptors different from those of d-endotoxins, is one facet of current strategies designed to delay resistance development. One of the interesting features of the Vip3A protein is that it shares no sequence homology with the known d- endotoxins [4]. Moreover, in ligand blotting experiments with brush border membrane vesicles (BBMV) from the tobacco hornworm, Manduca sexta, activated Cry1Ab and Vip3A bound to different molecules receptors [12]. These differences between Cry and Vip3 toxins of B. thuringi- ensis are very important to resolve the pest resistance problems. Prays oleae is a pest that causes a lot of damages to olive crops. In this report, we demonstrate that the insec- ticidal proteins Vip3LB and Cry1Ac bind to different L. Abdelkefi-Mesrati Á S. Rouis Á S. Sellami Á S. Jaoua (&) Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia e-mail: samir.jaoua@cbs.rnrt.tn Mol Biotechnol (2009) 43:15–19 DOI 10.1007/s12033-009-9178-4