The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates; (ii) Bt toxin directly and specifically binds glycolipids; and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.

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We reported previously a direct correlation between reduced soybean agglutinin binding to 63-and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens).In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [ 125 I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F 1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.Keywords: alkaline phosphatase; Cry1Ac; Heliothis virescens; resistance; N-acetylgalactosamine. Specific binding to insect midgut receptors is a key step in the mode of action of insecticidal Cry toxins from the bacterium Bacillus thuringiensis (Bt). Despite exceptions [1], in most cases Cry toxin specificity and potency correlate with the extent of toxin binding to midgut brush border membrane receptors in vitro [2,3]. Effective toxin binding to receptors results in toxin insertion and oligomerization on the midgut cell membrane, leading to pore formation and cell death by osmotic shock [4].In brush border membrane vesicles (BBMV) from Heliothis virescens (tobacco budworm) larvae, three groups of binding sites (A, B, and C) for Cry1A toxins were proposed based on their toxin binding specificities [5,6] Membrane-bound ALP from Bombyx mori and M. sexta are attached to the brush border cell membrane by a glycosylphosphatidylinositol (GPI) anchor [13][14][15]. Specific interactions between Cry1Ac and ALPs under native conditions resulting in inhibition of phosphatase activity have been reported for M. sexta [16] and H. virescens [17]. However, the potential role for alkaline phosphatases in Cry1Ac intoxication has not been addressed directly.The main goals of the present study were to identify the 68-kDa glycoprotein and characterize its oligosaccharide residues as a first step to investigate the specific alteration of this glycoprotein in Cry1Ac-resistant YHD2 larvae. Based on reported molecular sizes of insect alkaline phosphatases, and their interaction with Cry1 toxins, we hypothesized the 68-kDa glycoprotein to be a form of alkaline phosphatase. Immunoblotting and enzymatic activity experiments identified the 68-kDa protein as a GPI-anchored form of alkaline phosphatase we term HvALP (for H. virescens alkaline phosphatase). Ligand blots and glycosidase digestion Correspondence to M. J...

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.

Continued success of the most widely used MATERIALS AND METHODSInsects. We studied P. xylostella from 13 laboratory colonies derived from individuals collected at eight field sites in Hawaii (11,25). Larvae were fed cabbage foliage and colonies were maintained at 280C as described (25). The LAB-P colony, which was not exposed to B.t., served as the primary reference susceptible colony (26).We investigated the stability of resistance to B.t. in six laboratory colonies that were started from a field population (called NO) from Oahu, HI. The NO population had been treated repeatedly with B.t. in the field (11) and had developed moderate resistance to Dipel, a wettable powder formulation of a crystal-spore mixture of B.t. subspecies kurstaki (27,28). In the first laboratory-reared generation (F1), the LC50 (concentration required to kill 50% ofinsects tested) of NO larvae was about 25 times greater than the LC5o of larvae from the susceptible LAB-P strain (11). The NO colony was reared without exposure to B.t. for 3 generations, and then it was split into four colonies: NO-P, NO-Q, NO-R, and NO-U (23). NO-P, NO-Q, and NO-R were selected for additional resistance (see ref. 23 and description below) and then, as part of the present study, were reared without exposure to B.t. to examine the stability of extremely high resistance. To examine the stability of moderate resistance, NO-U was maintained without any additional exposure to insecticide for 35 generations. Results from the first 15 generations of rearing NO-U without exposure to B.t. were reported in detail previously (23) and are summarized here for comparison with results from NO-P, NO-Q, and NO-R. Selection and Reselection Experiments. NO-P, NO-Q, and NO-R were selected for additional resistance by feeding Abbreviations: B.t., Bacillus thuringiensis; ICP, insecticidal crystal protein; AI, active ingredient. tTo whom reprint requests should be addressed. 4120The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R1 is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and 1 H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of ␤-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (Kd ‫؍‬ 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)-and low (1 M)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope ( 1416 GVLTLNIQ 1423 ) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops.Cry insecticidal protein ͉ synergist B acillus thuringiensis (Bt) Cry1A proteins are pore-forming toxins that are specifically toxic to insect larvae in the order Lepidoptera. This family of proteins is widely used for insect control with Bt-transgenic crops, particularly cotton, and Bt microbial pesticides. Two issues on the deployment of Bt crops are the evolution of resistance in target pests and the lower level of control of specific target pests. Although insect resistance to Bt cotton has not caused a control failure in the field, there is a natural difference in lepidopteran susceptibility that effects insect control. For example, Bt cotton expressing only a Cry1A protein is highly effective in controlling tobacco budworm (Heliothis virescens) populations, whereas control of cotton bollworm (Helicoverpa zea) larvae is only achieved after additional insecticide treatment (1).The mode of action for Cry1A toxins includes sequential steps that determine their specificity. After ingestion by the lepidopteran larvae, Cry1A proteins are solubilized and activated to a toxic form by the insect digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins on the midgut microvilli. According to a current model (2), monomeric toxin binds a cadherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers display high-affinity binding to proteins that are attached to the cell membrane by a glycosylphosphatidylinositol anchor, such as aminopeptidase or alkaline phosphatase. This binding and t...