Introduction of a Lepidopteran-Specific Insecticidal Crystal Protein Gene of Bacillus thuringiensis subsp. kurstaki by Conjugal Transfer into a Bacillus megaterium Strain That Persists in the Cotton Phyllosphere (original) (raw)
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Applied and …, 1994
A lepidopteran toxin gene of the entomopathogen BaciUus thuringiensis subsp. kurstaki HD-1 was introduced into a cotton leaf-colonizing Bacilus megaterium strain, RS1, by conjugal transfer. Rifampinand nalidixic acid-resistant colonies obtained after cell mating were screened for crystal production by microscopy. A transcipient, B. megaterium RS1-43, was selected by this procedure. Southern blot hybridization with both total DNA and HindIII-digested DNA of the transcipient showed positive signals with a cryL4-specific probe, suggesting the transfer of the lepidopteran-specific cryL4(a) gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis confirmed the presence of the 134-kDa toxic crystal protein specific to lepidopteran larvae in the transcipient. Survival studies with cultures of the transcipient at both vegetative and postvegetative growth stages on cotton, under field conditions, suggested that the bacterium persisted on the leaf surfaces for more than 28 days, with a gradual decline in the population level with time, while the donor, B. thuringiensis subsp. kurstaki, disappeared completely after 7 days following inoculation. An in situ differential crystal-staining technique indicated the production of crystals by the transcipient on cotton leaf surfaces for about 30 days. Leaf bioassays of cotton plants inoculated with a single spray of the transcipient showed 75to 96% mortality to the first-instar larvae of Heliothis armigera up to 21 days, and this single spray conferred total protection to the plants for about 30 days by causing an antifeeding effect on the remaining larvae.
Journal of Economic Entomology, 2008
Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against Þve of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hü bner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with Cry1Ac. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S. frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30 Ð 80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S. frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20 Ð 69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d ßower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, ßower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves signiÞcantly expressed the highest, and large leaves, calyx, and bracts expressed signiÞcantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed signiÞcantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed signiÞcantly (P Ͻ 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.
Resistance: A Threat to the Insecticidal Crystal Proteins of Bacillus thuringiensis
The Florida Entomologist, 1995
Insecticidal crystal proteins (also known as δ-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the environment, and 4) highly amenable to genetic engineering. The use of transgenic plants expressing Bt δ-endotoxins has the potential to greatly reduce the environmental and health costs associated with the use of conventional insecticides. The complex mode of action of Bt is the subject of intensive research. When eaten by a susceptible insect δ-endotoxin crystals are solubilized in the midgut; proteases then cleave protoxin molecules into activated toxin which binds to receptors on the midgut brush border membrane. Part of the toxin molecule inserts into the membrane causing the midgut cells to leak, swell, and lyse; death results from bacterial septicemia. Insecticides formulated with Bt account for less than 1% of the total insecticides used each year worldwide because of high cost, narrow host range, and comparatively low efficacy. Environmental contamination, food safety concerns, and pest resistance to conventional insecticides have caused a steady increase in demand for Bt-based insecticides. The recent escalation of commercial interest in Bt has resulted in more persistent and efficacious formulations. For example, improved Bt-based insecticides have allowed management of the diamondback moth, Plutella xylostella (L.). Unfortunately this has resulted in the evolution of resistance to δ-endotoxins in P. xylostella populations worldwide. The recent appearance of Bt resistance in the field, corroborated by the results of laboratory selection experiments, demonstrates genetically-based resistance in several species of Lepidoptera, Diptera, and Coleoptera. The genetic capacity to evolve resistance to these toxins is probably Florida Entomologist 78(3) September, 1995 ducir la presión de seleccción, minimizando la exposición a Bt e incrementando otros factores de mortalidad, para disminuir la velocidad de adaptación de la plaga a Bt. The bacterium Bacillus thuringiensis Berliner (Bt) is a complex of subspecies characterized by their ability to synthesize crystalline inclusions during sporulation. These crystalline inclusions are comprised of relatively high quantities of one or more glycoproteins known as δ-endotoxins or Cry toxins (Table 1). The toxins produced by Bt play a vital role in the pathogenicity of this bacterium to insects and other invertebrates. The Cry toxins have enormous commercial value as safe, biodegradable pesticides. The specificity of Bt toxicity is highly desirable in integrated pest management (IPM) programs, particularly in sensitive aquatic and forest ecosystems where other life forms, including many beneficial and nontarget insects, must be conserved (May 1993). The selective toxicity, rapid environmental degradation, and vertebrate safety of Bt-based insecticides provide growers and the public with environmentally friendly and effective alternatives to conventional insecticides (Meadows 1993). Advances in biotechnology and genetic engineering, as well as the proteinaceous nature of the Cry toxins, led to the selection of the cry genes as the primary insect-resistance genes transferred into, and expressed in, plants and microbes (
The novel δ-endotoxin from Bacillus thuringiensis (Bt) is one of the alternative measures for lepidopteran pest control. A survey of 31 Bt isolates, obtained from Thailand and previously screened for their toxicity to lepidopterans, was conducted to determine the presence of cry1-type genes, using polymerase chain reaction -restriction fragment length polymorphism analysis. Seven distinct types of cry1 genes: cry1Aa, cry1B, cry1C, cry1Cb, cry1D, cry1E, and cry1F, were identified. The most common of the cry1-type genes was cry1Cb, followed by cry1C and cry1D, which covered 64.6, 48.4, and 25.8%, respectively. Besides cry1C and cry1Cb, two candidate cry genes, cry1E and cry1D, of isolate JC 190 (harbouring cry1C/1E), with cotton bollworm toxin specificity, showed 99% amino acid sequence identity to Cry1Ea of B. thuringiensis subsp. kenyae, while isolate JC 291 (containing cry1C/1Cb/1D), with Asian corn borer toxin specificity, harboured Cry1Dc, which exhibited only 84% amino acid sequ...
Gene, 1986
Cloning and expression of the lepidopteran toxin produced by ~aciZ~~s t~uringie~sis var. t~uri~gie~sis in Escherichia coli (Recombinant DNA; plasmid size determination; pACYC; restriction analysis; colony hybridization, bioassay) SUMMARY The BaciZlus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. th~r~ngiensjs plasmid pTN4 and cloning into the E. co/i pACYC 184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BumHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain bet-finer 17 15 has been shown to contain the gene that codes for the toxic protein of the crystal . No homologous sequences have been found between pTNG33 and the DNA of B. thu~ngie~is var. e~tomocidMs strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.
Journal of biochemistry and molecular biology, 2007
Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, wit...
Applied and environmental microbiology, 1988
We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pi...
This work has been carried out in order to construct a potent Bacillus thuringiensis (Bt) strain active against the Egyptian cotton leaf worm S. littoralis. Toward this target, the δ-endotoxin crystal protein genes cry1C (that encodes an insecticidal protein highly specific to S. littoralis) and cry1Ag (1Ac like) were used. HindIII digested cry1C was ligated into the HindIII site of the shuttle vector pHT7593 yielding the plasmid pHTNC3. HincII digested cry1Ag was ligated into SmaI site of pHT7593 and into SmaI site of pHTNC3. The three plasmid constructs were used to transform E. coli strain JM109. Colonies likely to contain these recombinant plasmids were screened for the production of toxin proteins. Positively identified transformants produced the expected size protein, detected by partial purification of protein, and is truncated upon trypsin digestion. The pHT7593-bearing cry1C, pHT7593-bearing cry1Ag and pHTNC3-harboring-cry1Ag were transferred into the non-crystalliferous (Cry-) Bt4 Bt strain. The introduction of the cry1Ag gene into Cry- Bt4 resulted in the formation of bipyramidal crystals. The introduction of both cry genes 1C and 1Ag resulted in the multiplication of bipyramidal crystals. In bioassays, cry1Ag-expressing BT4 Bt strain caused mortality of S. littoralis larvae only slightly (the LC50 was 104 ppm). In the presence of only Cry1C, the LC50 was 64 ppm. In presence of Cry1C co-expressed with Cry1Ag the LC50 decreased to 2.2 ppm. Thus, a combination of the Cry proteins 1C and 1Ag could result in effective insect control. With this approach, a combination of Cry proteins can be designed rather than discovered.
New Zealand Journal of Crop and Horticultural Science, 1992
The gene encoding the 133 kDa insecticidal crystal protein of Bacillus thuringiensis var. kurstaki isolate DSIR732, has been cloned into Escherichia coli. The complete nucleotide sequence of the coding region of the cry 732 gene, has been detennined and indicated a large open reading frame (ORF) of 3534 nucleotides encoding a protein of 1178 amino acid residues, with an estimated molecular weight of 133 330 Da. In addition, the sequence of 387 nucleotides 5' and 257 nucleotides 3' of the coding region has been detennined. Sequence comparisons of this cry 732 gene and its flanking regions with those of other Bt cry genes revealed that it is a member of the cry IA(c) class of B t cry genes. Alignment of the cry 732 nucleotide sequence with that of the cry IA(c) holotype gene sequence from B t kurstaki HD73 revealed only four nucleotide differences in the ORF, none of which resulted in amino acid substitutions, and one nucleotide difference in the region 5' to the ORF. Nucleotide sequence comparisons suggested that the cry 732 gene may have resulted from recombination events between the cry IA(c) genes of Bt kurstaki HD73 and Bt kenyae HD558-2. The cry 732 gene has been expressed in E. coli and bioassays, using extracts of E. coli producing the 133 kDa Cry732 protein, have demonstrated that the recombinant protein retains its insecticidal properties against three species of leafrollers (Lepidoptera: Tortricidae).
Biological Control, 2009
Among the pests of cultivated plants, the Spodoptera species complex is one of the most important. This genus is composed of polyphagous insects that attack, among other crops, cotton, corn, soy and castor bean. In Brazil Spodoptera frugiperda has been recognized as a primary pest in cotton, and Spodoptera eridania (Cramer) and Spodoptera cosmioides (Walker) species are important pests in cotton and soy. This study was performed in 2008 and was focused on selecting and characterizing Bacillus thuringiensis strains highly pathogenic to S. cosmioides, S. eridania and S. frugiperda and identifying Cry proteins toxic to these species. One hundred strains that exhibited toxicity to Lepidoptera were evaluated through bioassay and the most toxic ones were characterized through morphologic, biochemical and molecular analyses and LC 50 values were determined. Since the cry1Aa, cry1Ab, cry1Ac and cry2Aa genes were present in most of the toxic strains, recombinant Bt producing toxins encoded by representative genes from these families, were individually tested. The Cry proteins showed different levels of toxicity to the three Spodoptera species, with Cry1Aa and Cry1Ab the most toxic to S. cosmioides, Cry2Aa the most toxic to S. eridania and Cry1Aa, Cry1Ab and Cry2Aa most toxic to S. frugiperda. Cry1Ac, a component of some transgenic cotton varieties such as Bollgard I, extensively used in Brazil, presented low toxicity to the three species studied.