Development and commercial use of Bollgard cotton in the USA--early promises versus today's reality.

Bollgard cotton is the trademark given to a number of varieties of cotton bio-engineered to produce an insecticidal protein from Bacillus thuringiensis (Bt). When produced by the modified cotton plants, this protein controls certain lepidopterous cotton insect pests. Commercially available since 1996, these cotton varieties are purchased under a license agreement in which the growers pay a fee and agree to abide by the terms, which include a 1-year license to use the technology and agreement to participate in an insect resistance management program. Today, Bollgard cotton is grown on more than one-third of all cotton acreage in the USA. This product has reduced cotton production costs and insecticide use by providing an effective alternative to chemical insecticides for the control of tobacco budworm, Heliothis virescens; cotton bollworm, Helicoverpa zea; and pink bollworm, Pectinophora gossypiella. The specificity and safety profile of the Bt protein produced in planta in cotton was maintained. It has retained its selectivity for lepidopterous insects and lacks the characteristics found in potential allergenic proteins. Fiber quality, the agronomic characteristics of the plant and seed composition remain unchanged. New cotton technology is being developed to provide improved insect control and a wider spectrum of activity. These future products could further reduce insecticide use in the production of cotton, while maintaining the high level of safety and reliability that has been demonstrated by five seasons of Bollgard cotton use.

Genetically improved potatoes: protection from damage by Colorado potato beetles.

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

Modification of the coding sequence enhances plant expression of insect control protein genes.

Increased expression of the insect control protein genes of Bacillus thuringiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modified (3% nucleotide difference) and to fully modified (21% nucleotide difference) cryIA(b) and cryIA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified cryIA(b) gene had a 10-fold higher level of insect control protein and plants with the fully modified cryIA(b) had a 100-fold higher level of CryIA(b) protein compared with the wild-type gene. Similar results were obtained with the fully modified cryIA(c) gene in plants. Specific sequences of the partially modified cryIA(b) gene were analyzed for their ability to affect cryIA(b) gene expression in tobacco. The DNA sequence of a single region was identified as important to the improvement of plant expression of the cryIA(b) gene. The increased levels of cryIA(b) mRNA were not directly proportional to the increased levels of CryIA(b) protein in plants transformed with the modified cryIA(b) genes, indicating that the nucleotide sequence of these genes had an effect in improving their translational efficiency in plants.

Insect resistant cotton plants.

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli.

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads.

Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7). A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid. Transposition of IS50L-tox into the chromosome of P. fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element. A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase. The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species. Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads.

Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

Integration of the delta-endotoxin gene of Bacillus thuringiensis into the chromosome of root-colonizing strains of pseudomonads using Tn5.

The delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 was cloned into Tn5 and the resulting Tn5-tox element transposed from a vector plasmid into the chromosome of six corn-root-colonizing strains of Pseudomonas fluorescens and Agrobacterium radiobacter. Chromosomal integration of the tox gene maximized stability and minimized the potential for horizontal transfer of the tox gene to other bacterial species. Expression of the tox gene was demonstrated by Western blot analysis and by toxicity against larvae of the tobacco hornworm (Manduca sexta). The method described illustrates how a given gene can be stably integrated into the chromosome of diverse bacterial species.

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.