Altered Protoxin Activation by Midgut Enzymes from a Bacillus thuringiensis Resistant Strain of Plodia interpunctella

Oppert, Brenda; Kramer, Karl J.; Johnson, D. E.; MacIntosh, Susan; McGaughey, W. H.

doi:10.1006/bbrc.1994.1134

Cited by 119 publications

(67 citation statements)

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“…To prepare gut extract (GE), midguts from 10 late-fourth-instar S. frugiperda larvae, reared on artificial diet (Southland Products Inc., Lake Village, AR), were dissected, pooled, vortexed, and centrifuged at 13,000 ϫ g for 15 min at 4°C to separate gut extract from the solid materials (31). Supernatant was collected, filtered through a 0.22-m-pore-size filter, aliquoted, and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

“…The effect of SfCad or MsCad on Cry1Fa toxin stability in S. frugiperda GE was evaluated by incubating Cry1Fa toxin (100 ng) alone or Cry1Fa toxin that had been preincubated with 1 g cadherin fragments for 1 h with 1 l GE in a final volume of 20 l carbonate buffer (0.1 M Na 2 CO 3 , 0.25 M NaCl, pH 9.6) for 5, 10, or 20 min at 30°C. At the end of each time point, toxin proteolysis was terminated by heating the samples for 10 min at 100°C (31). Samples in SDS sample buffer were heated for 10 min at 100°C, separated by gel electrophoresis as described above, and electroblotted onto a PVDF membrane, and residual Cry1Fa toxin was detected using anti-Cry1Fa serum as described above.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to the pore formation model, the cell-signaling model (54) proposes that binding of activated toxin monomers to cadherin activates an intracellular signaling pathway, which ultimately results in cell death. However, toxin activation by gut proteases and toxin-receptor interactions are the most essential steps of the Bacillus thuringiensis mode of action and are common to all models, and alterations in these steps have been linked with resistance development (14,30,31,51,52).A strategy successfully employed to delay development of resistance against chemical pesticides is the use of synergists to enhance toxicity (3). Several synergists of Bacillus thuringiensis Cry that show low to moderate synergism of Cry toxicity have been reported.…”

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confidence: 99%

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Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

Rahman

Abdullah

Ambati

et al. 2012

Appl Environ Microbiol

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The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad-and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. Bacillus thuringiensis (Bt) Cry toxins are a family of bacterial pore-forming proteins that are highly toxic to a range of crop pests and disease vectors. Cry proteins are produced as crystals during the sporulation phase. Crystals are ingested, solubilized in the gut lumen to protoxin, and activated by host gut proteases. Activated toxin crosses the peritrophic matrix (PM) and binds cadherin, which is the primary high-affinity receptor for Cry1 toxins on the apical border of midgut microvilli. A current model postulates that toxin interaction with cadherin causes a conformational change in toxin allowing a specific proteolytic cleavage and formation of a prepore toxin oligomer. Evidence suggests that the prepore oligomer has increased affinity for secondary glycosylphosphatidylinositol (GPI)-anchored receptors, such as aminopeptidases (APNs) or alkaline phosphatases (ALPs) localized in lipid rafts. Oligomers insert into the membrane and disrupt membrane integrity by forming lytic pores, which lead directly to insect mortality or indirectly to mortality due to septicemia (5,7,21,45). A recent modification to the pore formation model described above proposes that activated toxin monomers first bind to abundant low-affinity APN receptors before binding to high-affinity cadherin receptors, which results in toxin oligomerization (32). In contrast to the pore formation model, the cell-signaling model (54) proposes that binding of activated toxin monomers to cadherin activates an intracellular signaling pathway, which ultimately results ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

Rahman

Abdullah

Ambati

et al. 2012

Appl Environ Microbiol

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“…Studies of insect resistance to Bt toxins have so far been mostly on Lepidoptera pests to the toxin Cry1Ab or Cry1Ac (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), the two major Bt Cry toxins which are highly toxic to Lepidoptera pests and known to share the same binding sites in target insects (4,20). Cry1Ab and Cry1Ac are the primary insecticidal proteins expressed in the current commercial transgenic Bt maize and Bt cotton varieties to target Lepidoptera pests in the field (21).…”

mentioning

confidence: 99%

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

Song

Kain

Cassidy

et al. 2015

Appl Environ Microbiol

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The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.T he Gram-positive soil bacterium Bacillus thuringiensis (Bt) has been widely used as a microbial insecticide in sprayable formulations, and Bt toxins are the primary insecticidal proteins expressed in genetically engineered crops to confer insect resistance (1; ISAAA's GM Approval Database, http://www.isaaa.org /gmapprovaldatabase/). Since the mid-1990s, insect-resistant Bt crops have been rapidly adopted with proven economic and environmental benefits (2, 3). However, development of insect resistance to Bt toxins threatens the long-term success of application of Bt toxins for insect pest control. The genetic potential of insect populations to evolve Bt resistance has been well shown in laboratory selections, and cases of insect resistance to Bt formulations and Bt crops have occurred in insect populations under selection pressure by Bt sprays and Bt crops in the field (4-8).Studies of insect resistance to Bt toxins have so far been mostly on Lepidoptera pests to the toxin Cry1Ab or Cry1Ac (9-19), the two major Bt Cry toxins which are highly toxic to Lepidoptera pests and known to share the same binding sites in target insects (4, 20). Cry1Ab and Cry1Ac are the primary insecticidal proteins expressed in the current commercial transgenic Bt maize and Bt cotton varieties to target Lepidoptera pests in the field (21). Resistance to Cry1Ac and Cry1Ab ...

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“…The specificity of the d-endotoxins has been postulated to be partly due to solubilization and proteolytic activation of toxin proteins in the insect midgut (Haider et al, 1986;Oppert et al, 1994), and to the presence of specific toxin receptors on the brush border membranes of midgut cells. Saturable, high-affinity binding sites for d-endotoxins have been described for some insect species (Hofmann et al, 1988a, b;van Rie et al, 1989;Knight et al, 1994Knight et al, , 1995Curtiss et al, 1995;Gill et al, 1995;Vadlamudi et al, 1995;Villa et al, 1995), and there is evidence that such binding initiates the toxins' lethal effects (van Rie et al, 1990;Ferre Â et al, 1991;MacIntosh et al, 1991;Sanchis et al, 1994;Masson et al, 1995a, b).…”

Section: Introductionmentioning

confidence: 99%

A Bacillus thuringiensis δ-endotoxin induces programmed cell death in mosquito larvae

Smouse

Nishiura

1997

Cell Death Differ

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We present evidence that a d-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae. After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling. The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death. Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei. The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells. This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis. From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.

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Altered Protoxin Activation by Midgut Enzymes from a Bacillus thuringiensis Resistant Strain of Plodia interpunctella

Cited by 119 publications

References 0 publications

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

A Bacillus thuringiensis δ-endotoxin induces programmed cell death in mosquito larvae

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