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Arthur I Aronson - One of the best experts on this subject based on the ideXlab platform.
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subspecies dependent regulation of bacillus thuringiensis Protoxin genes
Applied and Environmental Microbiology, 1999Co-Authors: Ping Cheng, Lan Wu, Yu Ziniu, Arthur I AronsonAbstract:Bacillus thuringiensis accumulates, primarily during sporulation, large quantities of insecticidal Protoxins which are deposited as crystalline, intracellular inclusions. Most subspecies contain several plasmid-encoded cry genes, each of which has a unique specificity. The overall toxicity profile of a subspecies depends not only on the array of cry genes present but also on the relative expression of the genes. In general, transcription depends on sporulation-specific sigma factors, but little is known about regulation of expression of the individual genes. In order to determine whether expression of a particular cry gene varies in different subspecies, lacZ fusions to the cry promoters of two Protoxin genes (cry1 class) were constructed. Protoxin accumulation and mRNA contents were also measured by performing immunoblotting and Northern analyses, respectively. The expression of a cry1Ab-lacZ fusion, but not the expression of a cry1C-lacZ fusion, was three to four times lower in B. thuringiensis subsp. aizawai strains than in B. thuringiensis subsp. kurstaki or B. thuringiensis subsp. tolworthi. Also, the Cry1Ab antigen and steady-state mRNA contents of B. thuringiensis subsp. aizawai were lower. The regulation of the genes must involve regions upstream of the promoters which are unique to each cry gene since (i) mutations in the upstream region of the cry1Ab gene resulted in enhanced expression in B. thuringiensis subsp. aizawai and (ii) no differences were found when the lacZ fusions contained the cry1Ab promoters but no upstream sequences. The capacity to regulate each of the Protoxin genes must be a factor in the overall Protoxin composition of a subspecies and thus its toxicity profile.
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specific binding of the e2 subunit of pyruvate dehydrogenase to the upstream region of bacillus thuringiensis Protoxin genes
Journal of Biological Chemistry, 1999Co-Authors: Thomas Walter, Arthur I AronsonAbstract:Abstract During sporulation, Bacillus thuringiensis produces inclusions comprised of different amounts of several related Protoxins, each with a unique specificity profile for insect larvae. A major class of these genes designatedcry1 have virtually identical dual overlapping promoters, but the upstream sequences differ. A gel retardation assay was used to purify a potential regulatory protein which bound with different affinities to these sequences in three cry1 genes. It was identified as the E2 subunit of pyruvate dehydrogenase. There was specific competition for binding by homologous gene sequences but not by pUC nor Bacillus subtilis DNA; calf thymus DNA competed at higher concentrations. The B. thuringiensis gene encoding E2 was cloned, and the purified glutathioneS-transferase-E2 fusion protein footprinted to a consensus binding sequence within an inverted repeat and to a potential bend region, both sites 200–300 base pairs upstream of the promoters. Mutations of these sites in the cry1A gene resulted in decreased binding of the E2 protein and altered kinetics of expression of a fusion of this regulatory region with the lacZ gene. Recruitment of the E2 subunit as a transcription factor could couple the change in post exponential catabolism to the initiation of Protoxin synthesis.
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the Protoxin composition of bacillus thuringiensis insecticidal inclusions affects solubility and toxicity
Applied and Environmental Microbiology, 1995Co-Authors: Arthur I AronsonAbstract:Most Bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid-encoded delta-endotoxin genes and package related Protoxins into a single inclusion. It was previously found that in B. thuringiensis subsp. aizawai HD133, which produces an inclusion comprising the CryIAb, CryIC, and CryID Protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mDa plasmid containing the cryIAb gene. As a result, inclusions produced by the cured strain were less readily solubilized at pH 9.2 or 9.5 and had a decreased toxicity for Plodia interpunctella, despite the presence of the CryIC Protoxin, which was active when solubilized. These results suggested that Protoxin composition was a factor in inclusion solubility and toxicity and that the cryIAb gene, which is also present on an unstable plasmid in several other subspecies, may have a unique role in inclusion solubility and toxicity. Introduction of a cloned copy of this gene into the plasmid-cured derivative of B. thuringiensis subsp. aizawai HD133 resulted in an increase in the solubility at pH 9.2 of all of the inclusion proteins from less than 20% to greater than 45% and a lowering of the 50% lethal concentration (LC50, in micrograms [dry weight] per square centimeter) of inclusions for Spodoptera frugiperda from 35 to 10. These values are the same as those found with inclusions from B. thuringiensis subsp. aizawai HD133, and in all cases, the LC50 of the solubilized Protoxins was 10. Transformants containing related cryIA genes produced inclusions which were more than 95% solubilized at pH 9.2 but also had LC50 of 10.(ABSTRACT TRUNCATED AT 250 WORDS)
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flexibility in the Protoxin composition of bacillus thuringiensis
Fems Microbiology Letters, 1994Co-Authors: Arthur I AronsonAbstract:In at least three Bacillus thuringiensis subspecies, multiple Protoxin genes are confined to just a few of the many plasmids with two or more on one of > 100 mDa and a particular gene, cryIA(b), on a 40–50 mDa plasmid. The latter is unstable but can be maintained in the population by cell mating. Cells which had lost this plasmid compensated by increasing transcription of the remaining Protoxin genes resulting in the formation of inclusions which differed from those in the parental strains in their toxicity profiles for selected insects as well as their solubility. Instability of a particular Protoxin-encoding plasmid appears to be a mechanism for rapidly shifting the Protoxin gene complement and thus the toxicity profiles of these bacteria.
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the solubility of inclusion proteins from bacillus thuringiensis is dependent upon Protoxin composition and is a factor in toxicity to insects
Applied and Environmental Microbiology, 1991Co-Authors: Arthur I Aronson, W Mcgaughey, D E JohnsonAbstract:Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three Protoxins, CryIA(b), CryIC, and CryID, and the CryIC Protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) Protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this Protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID Protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble Protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble Protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize Protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) Protoxin in the former. The interactions of structurally related Protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.
Brenda Oppert - One of the best experts on this subject based on the ideXlab platform.
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cis mediated down regulation of a trypsin gene associated with bt resistance in cotton bollworm
Scientific Reports, 2015Co-Authors: Yutao Xiao, Brenda Oppert, Bruce E Tabashnik, Xianchun Li, Kongming WuAbstract:Transgenic plants producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are useful for pest control, but their efficacy is reduced when pests evolve resistance. Here we examined the mechanism of resistance to Bt toxin Cry1Ac in the laboratory-selected LF5 strain of the cotton bollworm, Helicoverpa armigera. This strain had 110-fold resistance to Cry1Ac Protoxin and 39-fold resistance to Cry1Ac activated toxin. Evaluation of five trypsin genes revealed 99% reduced transcription of one trypsin gene (HaTryR) was associated with resistance. Silencing of this gene with RNA interference in susceptible larvae increased their survival on diets containing Cry1Ac. Bioassays of progeny from crosses revealed that resistance to Cry1Ac was genetically linked with HaTryR. We identified mutations in the promoter region of HaTryR in the resistant strain. In transfected insect cell lines, transcription was lower when driven by the resistant promoter compared with the susceptible promoter, implicating cis-mediated down-regulation of HaTryR transcription as a mechanism of resistance. The results suggest that H. armigera can adapt to Bt toxin Cry1Ac by decreased expression of trypsin. Because trypsin activation of Protoxin is a critical step in toxicity, transgenic plants with activated toxins rather than Protoxins might increase the durability of Bt crops.
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bacillus thuringiensis cry3aa Protoxin intoxication of tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2012Co-Authors: Brenda Oppert, A G Martynov, E N ElpidinaAbstract:Abstract The yellow mealworm, Tenebrio molitor , is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa Protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the Protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in Protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.
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characterization of cdnas encoding serine proteases and their transcriptional responses to cry1ab Protoxin in the gut of ostrinia nubilalis larvae
PLOS ONE, 2012Co-Authors: Lawrent L. Buschman, Brenda Oppert, Chitvan KhajuriaAbstract:Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) Protoxin activation and Protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab Protoxin from 2 to 24 h, whereas one trypsin (OnTry2) was down-regulated at all time points. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab Protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.
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efficacy of bacillus thuringiensis cry3aa Protoxin and protease inhibitors against coleopteran storage pests
Pest Management Science, 2011Co-Authors: Brenda Oppert, T D Morgan, Karl J KramerAbstract:BACKGROUND: Environmental impacts and resistance to insecticides pose serious challenges to stored-product insect and other types of pest control. Insect-resistant transgenic grain is a potential alternative to fumigants, but candidate control proteins are needed, especially for coleopterans. Therefore, we evaluated the efficacy of a coleopteran-active toxin, Bacillus thuringiensis Cry3Aa, with or without protease inhibitors, in laboratory feeding assays against coleopteran storage pests. RESULTS: In a comparison of the toxicity of Cry3Aa Protoxin towards three species of coleopteran storage pests, Tenebrio molitor L. was found to be most sensitive, Tribolium castaneum (Herbst.) was most refractory and Rhyzopertha dominica F. displayed an intermediate response. For R. dominica, Cry3Aa combined with 3500 mg potato carboxypeptidase inhibitor or 5000 mg aprotinin kg−1 diet resulted in both delayed development and increased mortality. Potato carboxypeptidase inhibitor and bovine aprotinin reduced the LC50 of Cry3Aa for R. dominica two- and threefold respectively. Cry3Aa treatment resulted in fewer progeny from R. dominica, and progeny was further reduced when the Protoxin was combined with potato carboxypeptidase inhibitor. CONCLUSIONS: These data support the hypothesis that a combination of Cry3Aa Protoxin and protease inhibitors, particularly a potato carboxypeptidase inhibitor, may have applications in control strategies for preventing damage to stored products and grains by coleopteran pests. Published 2011 by John Wiley & Sons, Ltd.
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screen of bacillus thuringiensis toxins for transgenic rice to control sesamia inferens and chilo suppressalis
Journal of Invertebrate Pathology, 2010Co-Authors: Yang Hu, Brenda Oppert, Qiang Fu, Jie Zhang, Yufa Peng, Zhitao ZhangAbstract:Abstract Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt Protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt Protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC 50 of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens , respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis . To evaluate interactions among Bt Protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All Protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis , with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt Protoxin mixtures were antagonistic in toxicity to S. inferens . In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high Protoxin concentrations. The data indicate the utility of Cry1Ca Protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.
Alejandra Bravo - One of the best experts on this subject based on the ideXlab platform.
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comprehensive analysis of cry1ac Protoxin activation mediated by midgut proteases in susceptible and resistant plutella xylostella l
Pesticide Biochemistry and Physiology, 2020Co-Authors: Lijun Gong, Alejandra Bravo, Mario Soberon, Shi Kang, Junlei Zhou, Youjun ZhangAbstract:Abstract Insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) have been widely used to control agricultural pests in both foliage sprays and transgenic crops. Nevertheless, rapid evolution of insect resistance to Cry toxins requires elucidation of the molecular mechanisms involved in Cry resistance. Two proposed models have been described to explain the toxicity of Cry proteins, the classic model states that Cry Protoxin is activated by midgut proteases resulting in activated toxin that binds to receptors and forms a pore in the midgut cells triggering larval death, and the newly proposed dual model of the mode of action of Bt Cry toxins states that Protoxin and activated toxins may have different mechanisms of action since several resistant strains to activated Cry toxins are still susceptible to the same Cry-Protoxin. Protoxin activation by midgut proteases is a key step in both models. Herein, we evaluated Cry1Ac Protoxin activation in a susceptible Plutella xylostella (L.) strain (DBM1Ac-S) and in the near-isogenic strain (NIL-R) with high field-evolved Cry1Ac resistance. Previous work showed that Cry1Ac resistance in NIL-R correlates with reduced binding to midgut receptors due to enhanced MAPK signaling pathway and down regulation of ABCC2 receptor. However, reduced midgut trypsin levels and altered midgut protease gene transcription were also observed in the Cry1Ac-resistant field isolated strain that is parent of the NIL-R strain. Therefore, we analyzed the midgut protease activities in both DBM1Ac-S and NIL-R strains. Detection of enzymatic activities showed that caseinolytic protease, trypsin and chymotrypsin activities were not significantly different between the susceptible and resistant strains. Furthermore, treatment with different trypsin or chymotrypsin inhibitors, such as Nα-tosyl- l -lysine chloromethyl ketone (TLCK) or Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not affect the susceptibility to Cry1Ac Protoxin of the DBM1Ac-S and NIL-R larvae. Bioassay results indicated that the NIL-R larvae showed similar resistant levels to both Cry1Ac Protoxin and trypsin-activated toxin. Taken together, our results demonstrated that high-level field-evolved Cry1Ac resistance in the NIL-R strain is independent of Cry1Ac Protoxin activation and the specific Protoxin mechanism of action. This discovery will strengthen our comprehensive understanding of the complex mechanistic basis of Bt resistance in different insects.
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reduced expression of a novel midgut trypsin gene involved in Protoxin activation correlates with cry1ac resistance in a laboratory selected strain of plutella xylostella l
Toxins, 2020Co-Authors: Lijun Gong, Shi Kang, Junlei Zhou, Fan Ye, Mazarin Akami, Qingjun Wu, Shaoli Wang, Baoyun Xu, Zhongxia Yang, Alejandra BravoAbstract:Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac Protoxin activation, thereby conferring higher resistance to Cry1Ac Protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac Protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac Protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.
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insect hsp90 chaperone assists bacillus thuringiensis cry toxicity by enhancing Protoxin binding to the receptor and by protecting Protoxin from gut protease degradation
Mbio, 2019Co-Authors: Blanca I Garciagomez, Alejandra Bravo, Sayra N Cano, Erika E Zagal, Edgar Dantangonzalez, Mario SoberonAbstract:ABSTRACT Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac Protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D Protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D Protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A Protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry Protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCEBacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A Protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins.
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the c terminal Protoxin region of bacillus thuringiensis cry1ab toxin has a functional role in binding to gpi anchored receptors in the insect midgut
Journal of Biological Chemistry, 2018Co-Authors: Arlen Penacardena, Ricardo Grande, Jorge Sanchez, Bruce E Tabashnik, Alejandra Bravo, Mario Soberon, Isabel GomezAbstract:: Bacillus thuringiensis Cry toxins are used worldwide for controlling insects. Cry1Ab is produced as a 130-kDa Protoxin that is activated by proteolytic removal of an inert 500 amino-acid-long C-terminal region, enabling the activated toxin to bind to insect midgut receptor proteins, leading to its membrane insertion and pore formation. It has been proposed that the C-terminal region is only involved in toxin crystallization, but its role in receptor binding is undefined. Here we show that the C-terminal region of Cry1Ab Protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) insect receptors. ELISA, ligand blot, surface plasmon resonance, and pulldown assays revealed that the Cry1Ab C-terminal region binds to both ALP and APN but not to cadherin. Thus, the C-terminal region provided a higher binding affinity of the Protoxin to the gut membrane that correlated with higher toxicity of Protoxin than activated toxin. Moreover, Cry1Ab domain II loop 2 or 3 mutations reduced binding of the Protoxin to cadherin but not to ALP or APN, supporting the idea that Protoxins have additional binding sites. These results imply that two different regions mediate the binding of Cry1Ab Protoxin to membrane receptors, one located in domain II-III of the toxin and another in its C-terminal region, suggesting an active role of the C-terminal Protoxin fragment in the mode of action of Cry toxins. These results suggest that future manipulations of the C-terminal Protoxin region could alter the specificity and increase the toxicity of B. thuringiensis proteins.
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dual mode of action of bt proteins Protoxin efficacy against resistant insects
Scientific Reports, 2015Co-Authors: Bruce E Tabashnik, Fangneng Huang, Jie Zhang, Yidong Wu, Alejandra Bravo, Min Zhang, Jeffrey A Fabrick, Alexander Yelich, Gopalan C Unnithan, Mario SoberonAbstract:Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive Protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac Protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than Protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which Protoxins and activated toxins kill insects via different pathways. Recognizing that Protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.
Luke Masson - One of the best experts on this subject based on the ideXlab platform.
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transcriptional response of choristoneura fumiferana to sublethal exposure of cry1ab Protoxin from bacillus thuringiensis
Insect Molecular Biology, 2006Co-Authors: Luke Masson, Roland Brousseau, L Meunier, Gabrielle Prefontaine, M Van MunsterAbstract:: Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal Protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal Protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and Protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after Protoxin exposure.
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Toxicity toSpodoptera exigua andTrichoplusia niofIndividual P1 Protoxins andSporulated Cultures ofBacillus thuringiensis subsp. kurstaki HD-1andNRD-12
1990Co-Authors: Luke Masson, Roland BrousseauAbstract:Thetoxicities toneonate Spodoptera exigua andTrichoplusia nioflyophilized powders obtained from sporulated liquid cultures (referred toassporulated cultures) andEscherichia coli-expressed P1[crylA(a) crylA(b) crylA(c)] Protoxins fromthree-gene strains ofNRD-12andHD-1ofBacillus thuringiensis subsp. kurstaki weredetermined byusing diet incorporation bioassays. Although sporulated cultures frombothstrains weremoretoxic toT.nithanS.exigua, there werenodifferences intoxicity between NRD-12andHD-1. Toxicities ofthethree individual P1Protoxins against S.exigua varied byatleast fivefold, withthecrylA(b) protein being themosttoxic. ThesesameProtoxins varied intoxicity against T.nibyatleast 16-fold, withthe cryIA(c) protein being themosttoxic. However, whentested against either S.exigua orT.ni,there wereno differences intoxicity between anNRD-12P1Protoxin andthecorresponding HD-1P1Protoxin. Comparing thetoxicities ofindividual Protoxins withthatofsporulated cultures demonstrates thatnoindividual Protoxin wasastoxic toS.exigua asthesporulated cultures. However, this samecomparison against T.nishowsthat boththecrylA(b) andcryIA(c) proteins areatleast astoxic asthesporulated cultures. Results fromthis study suggest thatNRD-12isnotmoretoxic toS.exigua thanHD-1,thatdifferent protein types havevariable host activity, andthatotherB.thuringiensis components arenotrequired forT.nitoxicity butthatother components suchasspores mightberequired forS.exigua toxicity. Although theinsecticidal bacterium Bacillus thuringiensis subsp. kurstaki (serotype H 3a:3b) HD-1caneffectively control someagriculturally important lepidopterous pests, suchasthecabbage looper (Trichoplusia ni[Hubner]), many keylepidopterous pests, suchasthebeetarmyworm (Spodoptera exigua[Hbfiner]), arerelatively tolerant and causeeconomic damage(14). Commercial formulations containing arelatively newstrain ofthis B.thuringiensis subspecies, NRD-12,wereshowntobethree tofour times more toxic toS.exigua thancommercial formulations ofHD-1 (13). Mostoftheinsecticidal activity against lepidoptera in B.thuringiensis subsp. kurstaki HD-1andNRD-12isattributedtotheP1crystal, whichiscomposed ofthree proteins belonging tothecryIA(a), cryIA(b), andcryIA(c) protein types(7,8,15). Moaretal.(15)reported thattheNRD-12 lyophilized powderobtained fromsporulated liquid cultures (referred tohereassporulated cultures) containing both proteinaceous crystals andspores was2.5-fold moretoxic against S.exigua thanwasHD-1.Mostofthis increased activity wasfoundtoreside intheP1crystal. However, subsequent studies revealed thattheHD-1strain usedin those tests wasmissing thecrylA(b) gene,whichhasbeen showntoberequired formaximumtoxicity toS.exigua (15, 19). Although these results demonstrated thatthecryIA(b) protein confers someinsecticidal activity, otherproteins alsomustbepartly responsible. Additionally, itisstill unclear whether NRD-12isinfactmoretoxic toS.exigua thanastrain ofHD-1containing allthree P1Protoxin genes and,ifso,whatfactor isresponsible forthis activity. Recently, thethreeP1Protoxin proteins ofHD-1and NRD-12havebeencloned andexpressed inEscherichia coli (L.Masson, G.Prefontaine, L.Peloquin, P.C.K.Lau,and
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Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 Protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12
Applied and Environmental Microbiology, 1990Co-Authors: W J Moar, Luke Masson, Roland Brousseau, J T TrumbleAbstract:The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] Protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 Protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same Protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 Protoxin and the corresponding HD-1 P1 Protoxin. Comparing the toxicities of individual Protoxins with that of sporulated cultures demonstrates that no individual Protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.
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comparative analysis of the individual Protoxin components in p1 crystals of bacillus thuringiensis subsp kurstaki isolates nrd 12 and hd 1
Biochemical Journal, 1990Co-Authors: Luke Masson, Gabrielle Prefontaine, L Peloquin, Roland BrousseauAbstract:Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa Protoxins (termed the 4.5, 5.3 and 6.6 Protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-Protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 Protoxins vary between the two crystals, whereas the 6.6 Protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.
Harvey Kaplan - One of the best experts on this subject based on the ideXlab platform.
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Bacillus thuringiensis crystal protein: effect of chemical modification of the cysteine and lysine residues.
Journal of Invertebrate Pathology, 2004Co-Authors: Christin T. Choma, Harvey KaplanAbstract:Abstract The 16 cysteine residues of reduced Protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl Protoxin; (b) iodoacetamide, giving carbaminomethyl Protoxin and (c) N-(β-iodoethyl)trifluoroacetamide to give aminoethyl Protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native Protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5–10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl Protoxin derivatives. Nonetheless, these succinylated Protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized Protoxin.
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Role of DNA in the Activation of the Cry1A Insecticidal Crystal Protein from Bacillus thuringiensis
Journal of Biological Chemistry, 1998Co-Authors: F R Clairmont, Ross E. Milne, Van Thong Pham, Marjolaine Carriere, Harvey KaplanAbstract:Abstract The Cry1A insecticidal crystal protein (Protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA. Only the N-terminal toxic moiety of the Protoxin was found to interact with the DNA. Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of Protoxin, indicating that only a small region of the N-terminal toxic moiety interacts with the DNA. It is proposed that the DNA-Protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward. It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the Protoxin. Activation of the Protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes. Larval trypsin initially converts the 20-kilobase pair DNA-Protoxin complex to a 20-kilobase pair DNA-toxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.
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Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA.
Journal of Biological Chemistry, 1993Co-Authors: H P Bietlot, Johann P. Schernthaner, Ross E. Milne, F R Clairmont, R S Bhella, Harvey KaplanAbstract:Abstract Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (Protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major Protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized Protoxin contained 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized Protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The Protoxin and DNA appeared to elute as a complex with a molecular mass of > 2 x 10(6) Da on gel-filtration chromatography. No toxin was generated from the Protoxin with trypsin after extensive digestion of the Protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.
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the toxic moiety of the bacillus thuringiensis Protoxin undergoes a conformational change upon activation
Biochemical and Biophysical Research Communications, 1991Co-Authors: Christin T. Choma, Witold K Surewicz, Harvey KaplanAbstract:Abstract Proteolytic processing of the 133-kDa crystal protein (Protoxin) from Bacillus thuringiensis subsp. kurstaki yields a 67-kDa insecticidal toxin. Differential scanning calorimetry was used to investigate whether the toxic moiety in the Protoxin molecule has the same conformation as activated toxin. Compared to Protoxin, toxin gives rise to a more complex endotherm which extends over a 10°C broader temperature range and contains a component occurring at a substantially higher temperature than any unfolding transition in the Protoxin endotherm. It is concluded that the toxic moiety undergoes a conformational change upon activation in which the thermal stability of at least one of its domains is significantly increased.
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folding and unfolding of the Protoxin from bacillus thuringiensis evidence that the toxic moiety is present in an active conformation
Biochemistry, 1990Co-Authors: Christin T. Choma, Harvey KaplanAbstract:The action of trypsin or papain on the 130-kDa crystal protein (Protoxin) from Bacillus thuringiensis subsp. kurstaki HD-73 yields a 67-kDa proteinase-resistant toxic fragment (toxin) which is derived from the N-terminal half of the molecule. Sensitivity to proteolysis and fluorescence emission spectroscopy showed that the toxin unfolded to a much greater extent in 6 M guanidinium chloride (GuHCl) than in 8 M urea. Protoxin also unfolded extensively in 6 M GuHCl, whereas in 8 M urea only the C-terminal half of the molecule had unfolded extensively. Both unfolded Protoxin and ufolded toxin refolded to their native and biologically active conformations. The biphasic unfolding observed for Protoxin suggests that the C-terminal half of the molecule unfolded rapidly, whereas the N-terminal toxic moiety unfolded at a much slower rate, similar to that of the free 67-kDa toxin. A 67-kDa fragment, derived from the N-terminal half of the molecule, could be generated from the Protoxin in the presence of either urea or GuHCl by treatment with proteinases. Compared to toxin in denaturants, this fragment was found to be more sensitive to proteolysis. However, on removal of the denaturants the fragment had the same proteinase resistance and cytolytic activity as native toxin. The increased proteinase sensitivity of the fragment generated in the presence of denaturants appears to be due to a perturbation in the conformation of the N-terminal toxic moiety. This perturbation is attributed to the unfolding of the C-terminal region of the Protoxin prior to its proteolysis to yield the 67-kDa fragment. It is concluded that unfolding and folding of the N-terminal half of the Protoxin molecule are essentially independent of the C-terminal half and that the proteolytic processing of Protoxin to toxin is not accompanied by a major conformational change in ethe toxic moiety
