RESUMEN

We previously identified a novel Heliothis virescens 110 kDa aminopeptidase N (APN) that binds Bacillus thuringiensis (Bt) Cry1Ac and Cry1Fa delta-endotoxins, and cloned an internal region of the 110 kDa APN gene (Banks et al., 2001). Here we describe the RACE-PCR cloning and sequence of a cDNA encoding 110 kDa APN. The 110 kDa APN gene was transiently co-expressed with green fluorescent protein (GFP) in Drosophila S2 cells using the pIZT expression vector. Enrichment of total membranes purified from S2 cells transfected with the 110 kDa APN gene had 3.3 fold increased APN enzymatic activity relative to enriched total membranes purified from S2 cells transfected with vector alone. Whereas the majority of S2 cells transfected with the 110 kDa APN gene bound rhodamine-labeled Cry1Ac toxin, no S2 cells transfected with vector alone bound rhodamine-labeled Cry1Ac toxin. This indicates that toxin binding to whole cells is APN mediated. However, flow cytometry and microscopy indicated that 110 kDa APN transfected S2 cells exposed to Cry1Ac or Cry1Fa toxin did not experience an increase in membrane permeability, indicating that APN transfected cells were resistant to toxin. This suggests while the H. virescens 110 kDa APN functions as a Bt toxin binding protein, it does not mediate cytotoxicity when expressed in S2 cells.

Asunto(s)

Antígenos CD13/genética , Mariposas Nocturnas/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD13/química , Antígenos CD13/metabolismo , Línea Celular , Clonación Molecular , Cartilla de ADN , Drosophila melanogaster , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD13/metabolismo , Metabolismo de los Hidratos de Carbono , Endotoxinas/metabolismo , Metabolismo de los Lípidos , Manduca/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Antígenos CD13/aislamiento & purificación , Cromatografía en Gel/métodos , Cromatografía en Capa Delgada/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Proteínas Hemolisinas , Marcaje Isotópico , Lípidos/aislamiento & purificación , Liposomas/metabolismo , Fosfatidilcolinas/metabolismo , Rubidio

RESUMEN

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.

Asunto(s)

Acetilgalactosamina/metabolismo , Aminopeptidasas/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Proteínas de Insectos/metabolismo , Lectinas de Plantas , Proteínas de Soja , Secuencia de Aminoácidos , Aminopeptidasas/aislamiento & purificación , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/inmunología , Biotina , Western Blotting , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endotoxinas/inmunología , Proteínas Hemolisinas , Proteínas de Insectos/aislamiento & purificación , Radioisótopos de Yodo , Lectinas/metabolismo , Ligandos , Datos de Secuencia Molecular , Mariposas Nocturnas , Ácido Peryódico , Reacción en Cadena de la Polimerasa/métodos , Dodecil Sulfato de Sodio , Coloración y Etiquetado/métodos

RESUMEN

The functional role of the alpha8 loop residues in domain II of Bacillus thuringiensis Cry1Ac toxin was examined. Alanine substitution mutations were introduced in the residues from 275 to 293. Among the mutant toxins, substitutions at R281 and R289 affected toxicity to Manduca sexta and Lymantria dispar. Loss of toxicity by these mutant toxins was well correlated with reductions in binding affinity for brush border membrane vesicles and the purified receptor, aminopeptidase N (APN), from both insects. These data suggest that the two arginine residues in the alpha8 loop region are important in toxicity and APN binding in L. dispar and M. sexta.

Asunto(s)

Aminopeptidasas/metabolismo , Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Proteínas de Insectos/metabolismo , Lepidópteros , Manduca , Sustitución de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Unión Competitiva/efectos de los fármacos , Bioensayo , Membrana Celular/química , Membrana Celular/metabolismo , Sistema Digestivo/química , Endotoxinas/genética , Endotoxinas/farmacología , Proteínas Hemolisinas , Larva , Microvellosidades/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Control Biológico de Vectores , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie

RESUMEN

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

Asunto(s)

Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Membrana Celular/metabolismo , Endotoxinas/metabolismo , Lepidópteros/metabolismo , Microvellosidades/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Sitios de Unión , Unión Competitiva , Técnicas Biosensibles , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Endotoxinas/toxicidad , Proteínas Hemolisinas , Immunoblotting , Ligandos , Resonancia por Plasmón de Superficie , Vesículas Transportadoras/metabolismo

RESUMEN

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Endotoxinas/química , Endotoxinas/metabolismo , Microvellosidades/metabolismo , Mariposas Nocturnas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/toxicidad , Sitios de Unión , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Endotoxinas/toxicidad , Proteínas Hemolisinas , Ligandos , Modelos Biológicos , Control Biológico de Vectores , Estructura Terciaria de Proteína

RESUMEN

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.

Asunto(s)

Antígenos CD13/química , Antígenos CD13/genética , Glicosilfosfatidilinositoles/química , Manduca/enzimología , Manduca/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD13/biosíntesis , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Expresión Génica , Técnicas In Vitro , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reticulocitos/metabolismo , Homología de Secuencia de Aminoácido , Spodoptera , Transfección

RESUMEN

Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD13/metabolismo , Endotoxinas , Acetilgalactosamina/metabolismo , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Sitios de Unión , Proteínas Hemolisinas , Ligandos , Manduca/metabolismo , Microvellosidades/metabolismo , Resonancia por Plasmón de Superficie

RESUMEN

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

RESUMEN

Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidad , Manduca/metabolismo , Acetilgalactosamina/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Unión Competitiva , Endotoxinas/química , Proteínas Hemolisinas , Microvellosidades/metabolismo , Modelos Moleculares , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Factores de Tiempo

RESUMEN

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

Asunto(s)

Fagos de Bacillus/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas , Endotoxinas/genética , Endotoxinas/toxicidad , Insecticidas , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/genética , Endotoxinas/química , Endotoxinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Vectores Genéticos , Proteínas Hemolisinas , Immunoblotting , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad

RESUMEN

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

Asunto(s)

Aminopeptidasas/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Proteínas de Insectos , Mariposas Nocturnas/enzimología , Receptores de Superficie Celular/metabolismo , Acetilgalactosamina/farmacología , Acetilglucosamina/farmacología , Amino Azúcares/farmacología , Aminopeptidasas/química , Aminopeptidasas/aislamiento & purificación , Animales , Toxinas de Bacillus thuringiensis , Glicosilfosfatidilinositoles/metabolismo , Proteínas Hemolisinas , Cinética , Rubidio/metabolismo , Rubidio/farmacología

RESUMEN

A purified, GPI-linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33-1.7 nM) than in receptor-free membranes. The non-toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CrylAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current-voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas , Endotoxinas/farmacología , Canales Iónicos/biosíntesis , Membrana Dobles de Lípidos/metabolismo , Manduca/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Antígenos CD13/metabolismo , Sistema Digestivo/enzimología , Sistema Digestivo/metabolismo , Proteínas Hemolisinas , Canales Iónicos/metabolismo

RESUMEN

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.

RESUMEN

The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3') was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer.

Asunto(s)

Arachis/genética , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/inmunología , Bioensayo , Southern Blotting , Células Cultivadas , Clonación Molecular , Endotoxinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Terapia Genética/métodos , Vectores Genéticos , Proteínas Hemolisinas , Higromicina B , Insectos , Mutagénesis Insercional , Proteínas de Plantas/inmunología , Plantas Modificadas Genéticamente , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Semillas/genética , Transformación Genética

RESUMEN

Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein. The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines. Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression. These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.

RESUMEN

Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene.

Asunto(s)

Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas , Endotoxinas/biosíntesis , Genes Sintéticos , Glycine max/fisiología , Transformación Genética , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Bioensayo , Cartilla de ADN , Endotoxinas/genética , Endotoxinas/toxicidad , Genes Bacterianos , Proteínas Hemolisinas , Lepidópteros , Control Biológico de Vectores , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Glycine max/genética

RESUMEN

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.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Proteínas de Insectos , Receptores de Superficie Celular/metabolismo , Acetilgalactosamina/farmacología , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Endotoxinas/aislamiento & purificación , Proteínas Hemolisinas , Cinética , Manduca , Peso Molecular , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo

RESUMEN

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

RESUMEN

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.