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PubMed1992

Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens.

Evolution of pest resistance to insecticidal proteins produced by Bacillus thuringiensis (Bt) would decrease our ability to control agricultural pests with genetically engineered crops designed to express genes coding for these proteins. Previous genetic and biochemical analyses of insect strains with resistance to Bt toxins indicate that (i) resistance is restricted to single groups of related Bt toxins, (ii) decreased toxin sensitivity is associated with changes in Bt-toxin binding to sites in brush-border membrane vesicles of the larval midgut, and (iii) resistance is inherited as a partially or fully recessive trait. If these three characteristics were common to all resistant insects, specific crop-variety deployment strategies could significantly diminish problems associated with resistance in field populations of pests. We present data on Bt-toxin resistance in Heliothis virescens, a major agricultural pest targeted for control with Bt-toxin-producing crops. A laboratory strain of H. virescens developed resistance in response to selection with the Bt toxin CryIA(c). In contrast to other cases of Bt-toxin resistance, this H. virescens strain exhibits cross-resistance to Bt toxins that differ significantly in structure and activity. Furthermore, the resistance in this strain is not accompanied by significant changes in toxin binding, and resistance is inherited as an additive trait when larvae are treated with high doses of CryIA(c) toxin. These findings have important implications for Bt-toxin-based pest control.

Journal Article↗

PubMed1996

Role of domain II, loop 2 residues of Bacillus thuringiensis CryIAb delta-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens.

Site-directed mutagenesis was used to examine the role of domain II, loop 2 residues, 368RRPFNIGI375, of Bacillus thuringiensis insecticidal protein CryIAb. Alanine substitution of residues 368RRP370, called B4, abolished potency toward Manduca sexta and Heliothis virescens, and the loss of toxicity was correlated directly to substantially reduced binding affinity to brush-border membrane vesicles (BBMV) prepared from the target insect midguts. These results indicated that these positive charges might be essential to orient the toxin to midgut receptor molecule(s). The role of residue Phe371 of CryIAb toxin to M. sexta was investigated by substituting a series of residues at this position. Irreversible binding and toxicity were affected significantly by hydrophilic, aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and Ser but not by Tyr or Trp. A hydrophobic aromatic side-chain residue at position 371 was therefore essential for irreversible binding of CryIAb toxin in M. sexta. The role of residues 370PFNIGI375 of CryIAb toxin on H. virescens was also examined. Mutants D2 (deletion of residues 370-375), G374A (alanine substitution of Gly374), and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.

Amino Acid Sequence↗

PubMed1999

Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel.

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.

Amino Acid Sequence↗

PubMed2000

Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide.

The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.

Amino Acid Sequence↗

PubMed2002

Directed molecular evolution: bridging the gap between genomics leads and commercial products.

Agricultural crops, engineered to express transgenic traits, have been rapidly adopted by farmers since the initial commercialization of this technology in 1996. However, despite nearly 20 years of research in agricultural biotechnology, only two product categories have achieved commercial success: plants containing transgenes conferring tolerance to herbicides and plants containing insecticidal protein genes derived from Bacillus thuringensis. A number of transgenic concepts, while exhibiting promising phenotypes in laboratory experiments, have failed to generate commercially viable crops. Many of the leads produced by modern integrative approaches to understanding plant biology will need further optimization to deliver economically viable crops. Directed molecular evolution represents a powerful technology to optimize newly discovered leads towards product objectives. In this review, we show by example how directed molecular evolution can be used to develop enabling technologies for plant biologists; how genes can be optimized to generate improved input traits such as those conferring insect tolerance, disease control and herbicide tolerance; and how plant quality can be altered to improve yield, produce novel industrial feedstocks and improve nutritional qualities.

Agriculture↗

PubMed1995

Direct effects of recombinant nuclear polyhedrosis viruses on selected nontarget organisms.

A limitation to effective field use of naturally occurring nuclear polyhedrosis viruses (NPVs) is the slow rate at which they kill their host. In making NPVs a more attractive pest management tool, this problem has been addressed by modifying NPVs genetically to express insecticidal proteins resulting in substantial increases in their speed of action. One concern associated with these recombinant NPVs, however, is their effects on nontarget insects associated with pests targeted for control by applications of NPVs. Our studies evaluated the direct effects of wild-type Autographa californica NPV (AcNPV) and a recombinant AcNPV (AcAaIT) on three insects beneficial to production agriculture. The recombinant NPV expresses an insect-selective neurotoxin, AaIT, which was isolated from the scorpion, Androctonus australis Hector. Two generalist predators, Chysoperla carnea Stephens and Orius insidiosus (Say), were not adversely affected by feeding on larvae of Heliothis virescens (F.) infected with AcAaIT. Similarly, no adverse effects were detected in the honey bee, Apis mellifera L., when injected with wild-type or recombinant NPVs. Results from this study may provide a foundation upon which potential risks associated with genetically engineered NPVs may be evaluated on a limited scale in greenhouse or field experiments.

Animals↗

PubMed2003

A spatially explicit model simulating western corn rootworm (Coleoptera: Chrysomelidae) adaptation to insect-resistant maize.

A stochastic spatially explicit computer model is described that simulates the adaptation by western corn rootworm, Diabrotica virgifera virgifera LeConte, to rootworm-resistance traits in maize. The model reflects the ecology of the rootworm in much of the corn belt of the United States. It includes functions for crop development, egg and larval mortality, adult emergence, mating, egg laying, mortality and dispersal, and alternative methods of rootworm control, to simulate the population dynamics of the rootworm. Adaptation to the resistance trait is assumed to be controlled by a monogenic diallelic locus, whereby the allele for adaptation varies from incompletely recessive to incompletely dominant, depending on the efficacy of the resistance trait. The model was used to compare the rate at which the adaptation allele spread through the population under different nonresistant maize refuge deployment scenarios, and under different levels of crop resistance. For a given refuge size, the model indicated that placing the nonresistant refuge in a block within a rootworm-resistant field would be likely to delay rootworm adaptation rather longer than planting the refuge in separate fields in varying locations. If a portion of the refuge were to be planted in the same fields or in-field blocks each year, rootworm adaptation would be delayed substantially. Rootworm adaptation rates are also predicted to be greatly affected by the level of crop resistance, because of the expectation of dependence of functional dominance on dose. If the dose of the insecticidal protein in the maize is sufficiently high to kill >90% of heterozygotes and approximately 100% of susceptible homozygotes, the trait is predicted to be much more durable than if the dose is lower. A partial sensitivity analysis showed that parameters relating to adult dispersal affected the rate of pest adaptation. Partial validation of the model was achieved by comparing output of the model with field data on population dynamics, and with field data documenting rootworm adaptation to cyclodienes and organophosphates.

Adaptation, Physiological↗

PubMed2003

Geographic variation in susceptibility of Chilo suppressalis (Lepidoptera: Pyralidae) to Bacillus thuringiensis toxins in China.

Geographic variation in the susceptibility of the striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), in China to Bacillus thuringiensis (Bt) insecticidal crystal proteins Cry1Ac and Cry1Ab was studied to establish baseline information for comparing the future response of populations with increased exposure to Bt products. Rice is the major host of C. suppressalis, and Bt rice ma) be released in China in the near future. Twelve populations of the pest were collected from the major rice-growing regions of China. LC50 estimates were determined for all populations for Cry1Ac and for eight populations for Cry1Ab. The bioassay results indicated that the range of LC50 in neonate larvae to Cry1Ac and Cry1Ab was from approximately 15 to approximately 157 mg (AI)/L and approximately 2 to approximately 34 mg (AI)/L, respectively. LC50 values were lower for Cry1Ab than for Cry1Ac, and there was a significant positive correlation between the two toxins tested.

Animals↗

PubMed2003

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud.

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.

Amino Acid Oxidoreductases↗

PubMed2005

Bt176 corn in poultry nutrition: physiological characteristics and fate of recombinant plant DNA in chickens.

A genetically modified Bt176 corn hybrid, which contains an insecticidal protein against the European corn borer, and its conventional, nonmodified counterpart were evaluated in 4 separate trials to verify substantial equivalence in feeding value and animal performance. Thirty-six individually kept laying hens and 3 replicates of 94 broiler chickens each, assigned to 12 cages, were fed 2 different hen and broiler diets containing either 60% conventional or 60% Bt176 corn. The nutrient compositions of the 2 corn hybrids and the 2 corn diets revealed no major differences. Furthermore, metabolism and performance data revealed no significant differences between the birds that received the conventional, nonmodified corn, and those that received the modified corn diets. The detection of the genetic modification, by PCR, in feed obtained from insect-resistant Bt corn, in tissues and products from animals fed Bt corn is described. In all evaluated chicken tissues of muscle, liver, and spleen, the corn-chloroplast ivr gene fragment was amplified. It can be deduced from these findings and from other studies that the transfer of DNA fragments into the body is a normal process that takes place constantly. Nevertheless, no recombinant plant DNA fragments such as recombinant bla or cry1A(b) fragments could be found. Bt-gene specific constructs from the Bt corn were not detected in any of the poultry samples, neither in organs, meat, nor eggs.

Animal Feed↗

PubMed1998

Premature polyadenylation at multiple sites within a Bacillus thuringiensis toxin gene-coding region.

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

Bacillus thuringiensis↗

PubMed2005

Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere.

Field studies were done to assess how much of the transgenic, insecticidal protein, Cry1Ab, encoded by a truncated cry1Ab gene from Bacillus thuringiensis (Bt), was released from Bt-maize MON810 into soil and whether bacterial communities inhabiting the rhizosphere of MON810 maize were different from those of the rhizosphere of nontransgenic maize cultivars. Bacterial community structure was investigated by SSCP (single-strand conformation polymorphism) of PCR-amplified 16S rRNA genes from community DNA. Using an improved extraction and detection protocol based on a commercially available ELISA, it was possible to detect Cry1Ab protein extracted from soils to a threshold concentration of 0.07 ng/g soil. From 100 ng of purified Cry1Ab protein added per gram of soil, only an average of 37% was extractable. At both field sites investigated, the amount of Cry1Ab protein in bulk soil of MON810 field plots was always lower than in the rhizosphere, the latter ranging from 0.1 to 10 ng/g soil. Immunoreactive Cry1Ab protein was also detected at 0.21 ng/g bulk soil 7 months after harvesting, i.e. in April of the following year. At this time, however, higher values were found in residues of leaves (21 ng/g) and of roots (183 ng/g), the latter corresponding to 12% of the Cry1Ab protein present in intact roots. A sampling 2 months later indicated further degradation of the protein. Despite the detection of Cry1Ab protein in the rhizosphere of MON810 maize, the bacterial community structure was less affected by the Cry1Ab protein than by other environmental factors, i.e. the age of the plants or field heterogeneities. The persistence of Cry1Ab protein emphasizes the importance of considering post-harvest effects on nontarget organisms.

Analysis of Variance↗

PubMed2000

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors.

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.

Bacillus anthracis↗

PubMed1991

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

Bacillus thuringiensis↗

PubMed1992

Evaluation of synergism among Bacillus thuringiensis toxins.

A simple test for synergism among toxins is described and applied to previously reported data on independent and joint toxicities of insecticidal proteins from Bacillus thuringiensis. The analysis shows synergism between a 27-kDa (CytA) toxin and 130- or 65-kDa (CryIV) toxins from B. thuringiensis subsp. israelensis against Aedes aegypti larvae. No positive synergism between 130- and 65-kDa toxins or among three CryIA toxins tested against seven species of Lepidoptera occurred. Comparisons with the original interpretations of these data show one case in which synergism occurred but was reported previously as absent and two cases that were not synergistic but were reported previously as suggestive of synergism. These results show that lack of an appropriate test for synergism can produce misleading conclusions. The methods described here can be used to test for synergistic effects of any poisons.

Animals↗

PubMed1992

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

Animals↗

PubMed1996

Characterization of a Novel Strain of Bacillus thuringiensis.

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.

Journal Article↗

PubMed1997

Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera).

The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.

Journal Article↗