Showing papers on "Bacillus thuringiensis published in 1994"
TL;DR: Insecticides derived from the common soil bacterium Bacillus thuringiensis (Bt) are becoming increasingly important for pest management as discussed by the authors, as mounting concerns about environmental hazards and widespread resistance in pest populations are reducing the value of conventional synthetic insecticides.
Abstract: Insecticides derived from the common soil bacterium Bacillus thuringiensis (Bt) are becoming increasingly important for pest management. Insecticidal crystal proteins (also called 8-endotoxins) from Bt are extremely toxic to certain pests, yet cause little or no hann to humans, most beneficial insects, and other nontarget organisms (19, 40). After proteolytic activation in the insect midgut, Bt toxins bind to the brush border membrane of the midgut epithelium and create pores that cause cells to swell and lyse (55). Technical innovations, including expression of Bt toxin genes in transgenic crop plants and transgenic bacteria, should increase the usefulness of Bt (6, 12, 35, 46, 91, 94, 109, 116). At the same time, mounting concerns about environmental hazards and widespread resistance in pest populations are reducing the value of conventional synthetic insecticides. Because Bt had been used commercially for more than two decades without reports of substantial resistance development in open field popu lations, some scientists had presumed that evolution of resistance was unlikely (21, 89). However, resistance to Bt was documented recently in field populations of diamondback moth in Hawaii, the continental US, and Asia (36, 42, 92a, 128, 129, 131, 138, 146, 148, 151). These reports confirmed suspicions raised by the results of laboratory selection for resistance to Bt in several major pests (102, 104, 135). Scientists in industry, government, and academia now recognize evolution of resistance to Bt in pests as the greatest threat to the continued success of Bt (18, 44, 58, 59, lOla, 108). To delay or reverse resistance to Bt in pests, we must first understand
981 citations
TL;DR: This chapter examines how the recently solved X-ray crystal structure of one Bt toxin allows to model the mode of action of a whole family of related protein toxins, and how a knowledge of the physiology of the insect targets of Bt toxins is needed for a better understanding of the toxic mechanism.
Abstract: Publisher Summary This chapter provides an overview of the biology of Bacillus thuringiensis ( Bt ). The chapter examines how the recently solved X-ray crystal structure of one Bt toxin allows to model the mode of action of a whole family of related protein toxins, and how a knowledge of the physiology of the insect targets of Bt toxins is needed for a better understanding of the toxic mechanism. Bacillus thuringiensis ( Bt ), a family of bacteria which make insecticidal proteins, accounts for 90–95% of the insect biocontrol market. This chapter illustrates that Bt is the name given to a family of bacteria found throughout the world. Paradoxically, CryIIIA, the only Bt toxin whose three-dimensional structure has been solved, kills very small insects, and its insolubility at neutral pH makes it difficult to assay in vitro . The crystal structure of CryIIIA permits site-directed mutagenesis and segment swapping experiments to be designed in a rational manner in order to identify the functional domains of the toxins. This chapter explores one attractive prospect that can be the replacement of the specificity domain with a binding domain directed to a target of choice, opening up the possibility of “designer” pesticides or a new class of immunotoxins. Characterization of toxin receptors and investigation of the mechanism of toxin synergism may assist in strategies for resistance management, a vital concern if Bt is to maintain its important position as the most extensively used biological insecticide.
375 citations
TL;DR: These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.
300 citations
TL;DR: Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance, and revertedant colonies had a higher proportion of extremely resistant individuals.
Abstract: Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.
252 citations
TL;DR: Neither PFGE nor MEE analysis could distinguish between the two species, which strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.
Abstract: Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.
226 citations
TL;DR: Simulation models suggest that wider at least some circumstances, transgenic plants bearing only one Bt gene may be more effective than sprays for delaying resistance to Bt.
Abstract: Insecticidal toxins from Bacillus thuringiensis (Bt) can now be deployed either in sprays or transgenic plants. Some entomologists and environmentalists have argued that the sprays are preferable to plants because they are less likely to cause resistance. However Bt sprays are not generally competitive with chemical insecticides and seem unlikely to displace them. In contrast, transgenic plants appear to be sufficiently effective to displace chemicals, making such plants attractive from the standpoint of environmental protection. Further, simulation models using data from the diamondback moth and a laboratory experiment using the Indianmeal moth suggest that wider at least some circumstances, transgenic plants bearing only one Bt gene may be more effective than sprays for delaying resistance to Bt Resistance in a laboratory‐selected strain of the Colorado potato beetle is especially interesting because a strain that can survive Bt sprays and develop to maturity cannot develop successfully on transgenic pl...
182 citations
Patent•
06 May 1994
TL;DR: An improved Bacillus thuringiensis (B.t.) delta-endotoxin was created by the modification of the gene encoding the toxin this paper. But the toxicity of a B.t. toxin was improved by replacing the native protoxin segment with an alternate protoxin fragment by constructing a chimeric toxin gene.
Abstract: An improved Bacillus thuringiensis (B.t.) delta-endotoxin is created by the modification of the gene encoding the toxin. The toxicity of a B.t. toxin was improved by replacing the native protoxin segment with an alternate protoxin segment by constructing a chimeric toxin gene.
176 citations
Patent•
11 Mar 1994
TL;DR: In this article, the authors describe methods useful for genetic engineering of plant cells to provide increased expression in the plastids of a plant or plant cell of the Bacillus thuringiensis insecticidal protein.
Abstract: Novel compositions and methods useful for genetic engineering of plant cells to provide increased expression in the plastids of a plant or plant cell of the Bacillus thuringiensis insecticidal protein.
175 citations
TL;DR: Fitness costs associated with resistance to conventional synthetic insecticides appear to vary considerably among species and insecticides, and little is known about fitness costs of resistance to insecticidal proteins from Bacillus thuringiensis, a common soil bacterium.
Abstract: Does adaptation to stress entail fitness costs in optimal environments? Antagonistic pleiotropy sometimes causes trade-offs in fitness across different environments in which the agent of stress is a pathogen (Lenski 1988) or a novel diet (Pashley 1988; Via 1991). It is commonly believed that evolution of resistance to insecticides also involves such trade-offs. Alleles conferring resistance are rare before exposure to insecticide and have been presumed to exert negative effects on fitness in the absence of insecticide (Crow 1957; Uyenoyama 1986; Hoffman and Parsons 1991). However, fitness costs associated with resistance to conventional synthetic insecticides appear to vary considerably among species and insecticides (Roush and McKenzie 1987; Roush and Daly 1990; Denholm and Rowland 1992). Moreover, little is known about fitness costs of resistance to insecticidal proteins from Bacillus thuringiensis, a common soil bacterium (McGaughey and Whalon 1992; Tabashnik 1994). Toxins from B. thuringiensis kill susceptible insects by binding to and disrupting the integrity of the midgut epithelium (Gill et al. 1992). Reduced binding of toxins is a primary mechanism of resistance in the diamondback moth, Plutella xylostella (Ferre et al. 1991), a major pest of cruciferous vegetables (Talekar 1992). The normal function of toxin-binding sites is not known but seems to be important because such sites are found in numerous insects (Feitelson et al. 1992). Several authors have suggested that alteration of toxin-binding sites may interfere with normal physiological functions (Van Rie et al. 1990; MacIntosh et al. 1991). Instability of resistance to B. thuringiensis in the absence of selection
158 citations
TL;DR: The first field trials in Australia of transformed cottons expressing the CryIA(b) insecticidal protein from Bacillus thuringiensis subsp.
Abstract: The first field trials in Australia of transformed cottons expressing the CryIA(b) insecticidal protein from Bacillus thuringiensis subsp. kurstaki (Bt) were completed during the 1992–93 season. The trials showed good efficacy of the plants against field populations of Helicoverpa, but there were indications of a declining level of Bt expression once plants began to senesce. Laboratory assays showed that larger instars could survive on the transgenic tissues although their growth was severely retarded. The introduction of Bt transgenic cottons may have several ecological impacts, apart from their direct impact on target pests. These include the risk of resistance development, effects on beneficial and non‐target arthropod species and changes in pest status associated with altered patterns of pesticide usage. Chief among the potential pests are sucking insects (e.g. Miridae) which appear not to be regulated by beneficial agents and are currently suppressed by sprays applied for Helicoverpa Transgenic Bt pl...
155 citations
TL;DR: A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology.
Abstract: A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects. Images
TL;DR: Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae, and a new protein with a broader activity was generated, suggesting its use as an alternative for CryIC in resistance management programs.
Abstract: To obtain Bacillus thuringiensis crystal proteins with new properties and to identify the regions involved in insecticidal activity, we generated hybrid genes composed of cryIC and cryIE by in vivo recombination. Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae. Transfer of this domain to CryIE, which is not active against these insects, resulted in a new protein with a broader activity. This hybrid protein binds to different receptors than CryIC, suggesting its use as an alternative for CryIC in resistance management programs.
TL;DR: The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.
TL;DR: The results suggest that the high toxicity of the wild‐type parasporal bodies of B. israelensis and morrisoni is due to synergism among three or four of their major proteins.
Abstract: Summary
The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio-assays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml−1, whereas the LC50 for the CryIVD was 85ng ml−1 In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.
Patent•
10 Aug 1994
TL;DR: A DNA which has the sequence shown in FIG. 5 and which defines a gene promoter region has been isolated from maize roots and used for driving expression of foreign genes in the roots of plants as discussed by the authors.
Abstract: A DNA which has the sequence shown in FIG. 5 and which defines a gene promoter region has been isolated from maize roots. The promoter may be used for driving expression of foreign genes in the roots of plants. This is particularly useful for expressing an insecticidal toxin, such as a delta-endotoxin of Bacillus thuringiensis, to impart resistance to insect attack on the roots of plants by Coleopteran insects.
TL;DR: Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, the cry- type genes of 225 Bacillus thuringiensis soil isolates from Taiwan were screened without much cost in time or labor and exhibited much greater activity against Plutella xylostella.
Abstract: Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities.
TL;DR: Functional data gathered from IS231 A inEscherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets, and a working model for DNA-protein interactions at the target site is proposed.
Abstract: Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of delta-endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 from Clostridium perfringens and distantly related to IS4 and IS186 from Escherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231A in Escherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained in Bacillus subtilis and B. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.
TL;DR: Alanine substitution (A3) or deletion of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type).
Abstract: Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type). The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B. mori brush border membrane vesicles. Some or all of the affected amino acid residues may interact directly or indirectly with the B. mori membrane receptor(s). Such receptor binding appears to be directly correlated with insect toxicity.
TL;DR: The data are consistent with the view that insecticidal crystals contain distorted, destabilized disulfide bonds which allow them to be solubilized at pH values characteristic of lepidopteran and dipteran larval midguts.
Abstract: We compared two insecticidal and eight noninsecticidal soil isolates of Bacillus thuringiensis with regard to the solubility of their proteinaceous crystals at alkaline pH values. The protein disulfide contents of the insecticidal and noninsecticidal crystals were equivalent. However, six of the noninsecticidal crystals were soluble only at pH values of ≥12. This lack of solubility contributed to their lack of toxicity. One crystal type which was soluble only at pH ≥12 (strain SHP 1-12) did exhibit significant toxicity to tobacco hornworm larvae when the crystals were presolubilized. In contrast, freshly prepared crystals from the highly insecticidal strain HD-1 were solubilized at pH 9.5 to 10.5, but when these crystals were denatured, by either 8 M urea or autoclave temperatures, they became nontoxic and were soluble only at pH values of ≥12. These changes in toxicity and solubility occurred even though the denatured HD-1 crystals were morphologically indistinguishable from native crystals. Our data are consistent with the view that insecticidal crystals contain distorted, destabilized disulfide bonds which allow them to be solubilized at pH values (9.5 to 10.5) characteristic of lepidopteran and dipteran larval midguts.
TL;DR: The expression of a cryIC-cryIA(b) fusion resulting in protection against S. exigua, H. virescens and M. sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies.
Abstract: Tobacco and tomato plants were generated exhibiting insect resistance due to the introduction of modified cryIA(b) and cryIC genes of Bacillus thuringiensis. Limited modifications at selected regions of the coding sequences of both genes are sufficient to obtain resistance against Spodoptera exigua, Heliothis virescens and Manduca sexta. The criteria used to modify both genes demonstrate that the removal of sequence motifs potentially resulting in premature polyadenylation and transcript instability causes increased insect resistance. The expression of a cryIC-cryIA(b) fusion resulting in protection against S. exigua, H. virescens and M. sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies.
TL;DR: Feeding assays of T1 plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance.
Abstract: Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T1 plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T1 plants correlated with the presence of the transgene.
TL;DR: Significant declines in realized h2 of resistance during individual selection experiments suggest that the initial frequency of resistance alleles was much higher than previously assumed, and rapid evolution of resistance to the mixture of HD-1 + HD-133, which contained at least six different toxins, contradicts the claim that multiple toxins prevent or greatly retard resistance development.
Abstract: Criteria for comparing the risk of resistance development among single insecticides and between mixtures and sequences of two insecticides are described. The rate of development of resistance to an insecticide is proportional to the population’s heritability (h2) of resistance to that insecticide. When cross-resistance is absent, a sequence of two insecticides is expected to be more durable than a mixture unless the population’s h2 of resistance to the mixture is less than half of the mean of the population’s h2 of resistance to the two individual components of the mixture. We applied these criteria to II previously reported selection experiments with the biopesticide Bacillus thuringiensis and Indianmeal moth, Plodia interpunctella (Hubner), a major pest of stored grain. The risk of resistance development did not differ significantly between the HD-1 strain of B. thuringiensis ssp. kurstaki and three other strains (HD-112, HD-133, and HD-198) of B. thuringiensis . Significant declines in realized h2 of resistance during individual selection experiments suggest that the initial frequency of resistance alleles was much higher than previously assumed. Our analysis also suggests that a mixture of HD-1 + HD-133 would not slow resistance development compared with a sequence of HD-1 followed by HD-133. Rapid evolution of resistance to the mixture of HD-1 + HD-133, which contained at least six different toxins, contradicts the claim that multiple toxins prevent or greatly retard resistance development.
TL;DR: The correlation of ribotype patterns with serotype suggests a clonal population structure for B. thuringiensis, and variety tenebrionis, a coleopteran pathogen, could be distinguished from the more common lepidopteran pathogens of this serotype by ribotyping.
Abstract: Ribosomal RNA gene restriction patterns have been determined for 43 strains of Bacillus thuringiensis representing 10 serovars and eight reference strains of B. anthracis, B. cereus and B. mycoides. Strains within a B. thuringiensis serovar produced highly related or identical ribotype patterns: in particular, 12 strains of serovar israelensis, five strains of serovar kurstaki, two strains of serovar galleriae and three strains of serovar aizawa produced ribotype patterns consistent with serotype designations. Moreover, variety tenebrionis (serotype 8a8b), a coleopteran pathogen, could be distinguished from the more common lepidopteran pathogens of this serotype (serovar morrisoni) by ribotyping. The correlation of ribotype patterns with serotype suggests a clonal population structure for B. thuringiensis.
TL;DR: The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of B. thuringiensis.
Abstract: Selection with Bacillus thuringiensis subsp. kurstaki, which contains CryIA and CryII toxins, caused a >200-fold cross-resistance to CryIF toxin from B. thuringiensis subsp. aizawai in the diamondback moth, Plutella xylostella. CryIE was not toxic, but CryIB was highly toxic to both selected and unselected larvae. The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of B. thuringiensis.
TL;DR: The results of this survey indicate that B. thuringiensis is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of B.ThuringiensIS isolates that may be used to control stored-product pests.
Abstract: Bacillus thuringiensis was isolated from dried tobacco residues and dead tobacco beetles (Lasioderma serricorne (F.); Coleoptera: Anobiidae) collected in a large number of locations worldwide. Eighty-eight samples of stored tobacco were analyzed and yielded 78 B. thuringiensis strains which were characterized on the basis of parasporal crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, and the results of an immunoblot analysis of the insecticidal crystal proteins. Flagellar antigen identification was used to differentiate selected isolates. Strains that produced rhomboidal crystals associated with the Coleoptera-specific pathotype (Cry III group) were the most abundant strains (59% of the isolates). Preliminary toxicity assays were performed with L. serricorne larvae, and the results suggested that activity is not restricted to isolates related to the Coleoptera-specific group. The results of our survey indicate that B. thuringiensis is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of B. thuringiensis isolates that may be used to control stored-product pests.
TL;DR: Results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.
Abstract: Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin. Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli. More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay. A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae. The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay. Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells. For the first loop, the differential specificity was not restricted to any single residue. In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype. The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants. All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop. These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation. These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.
TL;DR: In this article, a bacterial endophyte was engineered for insecticidal activity against the European corn borer by using an integrative plasmid vector, and the integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. cynodontis.
Abstract: A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The cryIA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryIA(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryIA(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. Images
TL;DR: Bt is now the accepted name for a range of aerobic spore-forming bacteria which form an insect toxic crystal during sporulation and many bacteriologists consider Bt to be a variant of Bacillus cereus, a ubiquitous soil-inhabiting bacterium.
TL;DR: Investigations of the function of the CytB C-terminal ‘tail’ showed that this δ-endotoxin is highly expressed and forms inclusions in an acrystalliferous Bt mutant without the aid of the 20 kDa ‘helper’ protein from Bt subspecies israelensis which is essential for CytA inclusion formation.
Abstract: The entomocidal δ-endotoxins CytA and CytB produced by Bacillus thuringiensis (Bt) subspecies israelensis and kyushuensis respectively showed a similar level of toxicity to mosquito larvae but were not toxic to the larvae of the lepidopteran Manduca sexta. CytA and CytB are also similar in sequence, predicted secondary structure and α-helical content, the only obvious difference being a C-terminal fifteen residue ‘tail’ on CytB. Investigations of the function, if any, of the CytB C-terminal ‘tail’ showed that this δ-endotoxin is highly expressed and forms inclusions in an acrystalliferous Bt mutant without the aid of the 20 kDa ‘helper’ protein from Bt subspecies israelensis which is essential for CytA inclusion formation. After proteinase K treatment, CytA and CytB were processed to virtually the same points in a sequence alignment and were equally haemolytic in vitro. However, the results suggested that unprocessed CytB differs from unprocessed CytA in that the former is not haemolytic.
TL;DR: Several lines of evidence for a complex genetic basis for resistance in tobacco budworm Heliothis virescens and diamondback moth Plutella xylostella are reviewed, suggesting that a genetic approach based on linkage mapping, capable of simultaneously analyzing multiple physiological mechanisms, will be very useful in dissecting the complex trait of resistance.
Abstract: Recent studies have shown that the genetic basis of resistance to Bacillus thuringiensis (Bt) toxin in insects is far from simple. In this respect, it is similar to resistance to chemical insecticides, which is conferred by multiple physiological mechanisms under independent genetic control. Several lines of evidence for a complex genetic basis for resistance in tobacco budworm Heliothis virescens and diamondback moth Plutella xylostella are reviewed. This suggests that a genetic approach based on linkage mapping, capable of simultaneously analyzing multiple physiological mechanisms, will be very useful in dissecting the complex trait of resistance. In Heliothis, the existence of separate, independently assorting resistance genes has already been confirmed by linkage analysis with marker loci. This approach assists in the measurement of the relative potency of different resistance genes present in the same strain. Additionally, it facilitates comparative studies of the independent acquisition of homologou...