Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects.

Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167-272 and amino acids 689-741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin.

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

Screening and identification of vip genes in Bacillus thuringiensis strains.

AIMS: To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis. METHODS AND RESULTS: Following a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48.9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains. CONCLUSIONS: Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that vip1 and vip2 genes have been identified by PCR-RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.

Detection and traceability of genetically modified organisms in the food production chain.

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.

Effect of Bacillus thuringiensis Cry1 toxins in insect hemolymph and their neurotoxicity in brain cells of Lymantria dispar.

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 microg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 microg/0.2 g fresh wt). Cry1E and Cry1Ac (5 microg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 microg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 microg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.

Bacillus thuringiensis and its use in transgenic insect control technologies.

The insecticidal activity of Bacillus thuringiensis (Bt) is mainly due to the production of crystals containing insecticidal crystal proteins (ICPs). These proteins are very selectively active against certain insect species, including some agronomically important pest species. Some ICP genes have been used for bioengineered crop protection, resulting in transgenic crop plants with excellent insect protection.

Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.

The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.

Bacillus thuringiensis and its pesticidal crystal proteins.

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis.

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.

Cloning and characterization of Manduca sexta and Plutella xylostella midgut aminopeptidase N enzymes related to Bacillus thuringiensis toxin-binding proteins.

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stringency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N (P. xylostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.

Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A delta-endotoxin may facilitate its coleopteran toxicity.

The Cry3A delta-endotoxin protein inclusion synthesized by Bacillus thuringiensis subsp. tenebrionis is soluble in alkaline and acid buffer solutions but the toxin precipitates when returned to neutral pH conditions. The midgut pH of susceptible beetle larvae is neutral to slightly acidic, a pH environment in which the Cry3A toxin is insoluble. To investigate this paradox we studied the Cry3A toxin after various proteolytic treatments. In many cases the toxin was cleaved into polypeptides that remained associated under non-denaturing conditions. Interestingly a chymotrypsinized Cry3A product was soluble under neutral pH conditions, retained full activity against susceptible beetle larvae, and exhibited specific binding to Leptinotarsa decemlineata midgut membranes.

Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella).

A colony of Plutella xylostella from crucifer fields in Florida was used in mortality bioassays with HD-1 spore, CryIA(a), CryIA(b), CryIA(c), CryIB, CryIC, CryID, CryIE, or CryIIA. The data revealed high levels of field-evolved resistance to HD-1 spore and all CryIA protoxins and no resistance to CryIB, CryIC, or CryID. CryIE and CryIIA were essentially not toxic. When HD-1 spore was combined 1:1 with protoxin and fed to susceptible larvae, spore synergized the activity of CryIA and CryIC 5- to 8-fold and 1.7-fold, respectively, and did not synergize the mortality of CryIIA. When fed to Florida larvae, spore failed to synergize the activity of all three CryIA protoxins, synergized the activity of CryIC 5.3-fold, and did not synergize the mortality for CryIIA. Binding studies with CryIA(b), CryIB, and CryIC were performed to determine possible mechanisms of resistance. The two techniques used were (i) binding of biotinylated toxin to tissue sections of larval midguts and (ii) binding of biotinylated toxin to brush border membrane vesicles prepared from whole larvae. Both showed dramatically reduced binding of CryIA(b) in resistant larvae compared with that in susceptible larvae but no differences in binding of CryIB or CryIC.

A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae.

The full characterization of a novel insecticidal crystal protein, named Cry9Ca1 according to the revised nomenclature for Cry proteins, from Bacillus thuringiensis serovar tolworthi is reported. The crystal protein has 1,157 amino acids and a molecular mass of 129.8 kDa. It has the typical features of the Lepidoptera-active crystal proteins such as five conserved sequence blocks. Also, it is truncated upon trypsin digestion to a toxic fragment of 68.7 kDa by removal of 43 amino acids at the N terminus and the complete C-terminal half after conserved sequence block 5. The 68.7-kDa fragment is further degraded to a nontoxic 55-kDa fragment. The crystal protein has a fairly broad spectrum of activity against lepidopteran insects, including members of the families Pyralidae, Plutellidae, Sphingidae, and Noctuidae. A 50% lethal concentration of less than 100 ng/cm2 of diet agar was found for diamondback moth, European corn borer, cotton bollworm, and beet armyworm. It is the first insecticidal crystal protein with activity against cutworms. No activity was observed against some beetles, such as Colorado potato beetle. The protein recognizes a receptor different from that recognized by Cry1Ab5 in Ostrinia nubilalis and Plutella xylostella. In Spodoptera exigua and P. xylostella, it binds to a receptor which is also recognized by Cry1Cax but with a lower affinity. In these insects, Cry1Cax probably binds with a higher affinity to an additional receptor which is not recognized by Cry9Ca1. Elimination of a trypsin cleavage site which is responsible for the degradation to a nontoxic fragment did result in protease resistance but not in increased toxicity against O. nubilalis.

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae).

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins.

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.

The C-terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding.

The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain-function relationships of the toxic fragment, hybrid crystal proteins based on CryIA(b) and CryIC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both dispalyed an insecticidal spectrum similar to that of the parental crystal protein from which the C-terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C-terminal part of the toxic fragment in receptor binding. These results demonstrate that the C-terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor.

The biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryIC] were compared between the field population and a laboratory strain. The field population proved resistant (greater than 200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brush-border membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.

Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins.

To investigate the biochemical basis of the differences in the insecticidal spectrum of Bacillus thuringiensis insecticidal crystal proteins (ICPs), we performed membrane binding and toxicity assays with three different ICPs and three lepidopteran species. The three ICPs have different toxicity patterns in the three selected target species. Binding studies with these 125I-labeled ICPs revealed high-affinity saturable binding to brush border membrane vesicles of the sensitive species. ICPs with no toxicity against a given species did not bind saturably to vesicles of that species. Together with previous data that showed a correlation between toxicity and ICP binding, our data support the statement that differences in midgut ICP receptors are a major determinant of differences in the insecticidal spectrum of the entire lepidopteran-specific ICP family. Receptor site heterogeneity in the insect midgut occurs frequently and results in sensitivity to more than one type of ICP.

Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis.

Receptor binding studies show that resistance of a laboratory-selected Plodia interpunctella strain to a Bacillus thuringiensis insecticidal crystal protein (ICP) is correlated with a 50-fold reduction in affinity of the membrane receptor for this protein. The strain is sensitive to a second type of ICP that apparently recognizes a different receptor. Understanding the mechanism of resistance will provide strategies to prevent or delay resistance and hence prolong the usefulness of B. thuringiensis ICPs as environmentally safe insecticides.

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects.

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.