0000000000023451
AUTHOR
M. Dolores Real
Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle.
Abstract Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when comp…
Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins.
ABSTRACT Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the …
IDENTIFICATION OF A CALMODULIN-BINDING SITE WITHIN THE DOMAIN I OF BACILLUS THURINGIENSISCry3Aa TOXIN
Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown that Cry3Aa toxin is proteolytically cleaved by CPB midgut membrane associated metalloproteases and that this cleavage is inhibited by ADAM metalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAM shedding substrates. In pull-down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaM in a calcium-independent manner. Furthermore, we used gel shift assays and (1)H NMR spectra to demons…
Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity
In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii…
Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum*
Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that th…
Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.
In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced u…
Occurrence of three different binding sites forBacillus thuringiensisδ-endotoxins in the midgut brush border membrane of the potato tuber moth,phthorimaea operculella(zeller)
The potato tuber moth is susceptible to at least three insecticidal crystal proteins (ICPs) from Bacillus thuringiensis: CrylA(b), CrylB, and CrylC. To design useful combinations of toxin genes either in transgenic plants or in new genetically modified B. thuringiensis strains, it is necessary to determine the binding characteristics of the different ICPs so as not to combine a pair sharing the same binding site. This has been accomplished using two different techniques: 125I-labeling of the ICPs with further measurement of the radioactivity bound to brush border membrane vesicles, and microscopic visualization of the bound ICPs by enzyme-linked reagents such as antibodies or streptavidin u…
Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin
When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to h…
Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster.
Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, “drosopterins,” and biopterin as final products. The regulatory relationship among the …
Analysis of kynurenine transaminase activity in Drosophila by high performance liquid chromatography
Abstract A sensitive assay for kynurenine transaminase activity (E.C. 2.6.1.7) based on rapid separation of the reaction product by high performance liquid chromatography (HPLC) has been developed. Drosophila sordidula extracts have been assayed by this new method and this is the first time that kynurenine transaminase activity has been demonstrated in Drosophila . The method of assay developed can be extended to any other organism. Kynurenine and 3-hydroxykynurenine were both used as substrates, and they were transaminated to kynurenic acid and xanthruenic acid, respectively. HPLC is used to separate and quantitate these reaction products from all other components in the reaction mixture. …
Unveiling gene expression regulation of the Bacillus thuringiensis Cry3Aa toxin receptor ADAM10 by the potato dietary miR171c in Colorado potato beetle
Background The Colorado potato beetle (CPB) is a worldwide devastating pest of potato plants and other Solanaceae characterized by its remarkable ability to evolve resistance to insecticides. Bacillus thuringiensis (Bt) Cry3Aa toxin represents an environmentally-safe alternative for CPB control but larvae susceptibility to this toxin has been reported to vary depending on the host plant on which larvae feed. To gain more insight into how nutrition mediates Bt tolerance through effects on gene expression here we explored the post-transcriptional regulation by miRNAs of the CPB-ADAM10 gene encoding the Cry3Aa toxin functional receptor ADAM10. Results The lower CPB-ADAM10 gene expression in CP…
The Histone Marks Signature in Exonic and Intronic Regions Is Relevant in Early Response of Tomato Genes to Botrytis cinerea and in miRNA Regulation
Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modific…
Identification of Stress Associated microRNAs in Solanum lycopersicum by High-Throughput Sequencing
Tomato (Solanum lycopersicum) is one of the most important crops around the world and also a model plant to study response to stress. High-throughput sequencing was used to analyse the microRNA (miRNA) profile of tomato plants undergoing five biotic and abiotic stress conditions (drought, heat, P. syringae infection, B. cinerea infection, and herbivore insect attack with Leptinotarsa decemlineata larvae) and one chemical treatment with a plant defence inducer, hexanoic acid. We identified 104 conserved miRNAs belonging to 37 families and we predicted 61 novel tomato miRNAs. Among those 165 miRNAs, 41 were stress-responsive. Reverse transcription quantitative PCR (RT-qPCR) was used to valida…
Inheritance of resistance to aBacillus thuringiensistoxin in a field population of diamondback moth (Plutella xylostella)
Inheritance of resistance to the Bacillus thuringiensis Berl. CryIA(b) crystal protein was studied in Plutella xylostella L. (diamondback moth). A field population 50-fold more resistant to CryIA(b) than a control susceptible strain was used. Dose-mortality curves of the resistant population, the susceptible strain and the F 1 from the two reciprocal crosses were compared. Resistance transmission to the F 1 was dependent on the sex of the resistant progenitor. Sex ratio of the survivors to high doses of CryIA(b) in the F 1 of the two reciprocal crosses did not corroborate the preliminary hypothesis of resistance being due to a recessive sex-linked allele. Since, in a previous work, the loss…
Proteome response of Tribolium castaneum larvae to Bacillus thuringiensis toxin producing strains.
Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC(50) values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major cat…
Proteomic insights into the immune response of the Colorado potato beetle larvae challenged with Bacillus thuringiensis.
Bacillus thuringiensis (Bt) toxins constitute effective, environmentally safe biopesticides. Nevertheless, insects' tolerance to Bt is influenced by environmental factors affecting immunity. To understand larval immune response in the devastating coleopteran insect pest Colorado potato beetle (CPB), we undertook a proteomic analysis of hemolymph of non-treated control larvae and larvae consuming non-lethal doses of spore-crystal mixtures containing the coleopteran-active Cry3Aa toxin. Results revealed lower amount of proteins involved in insect growth and higher amount of immune response-related proteins in challenged insects, sustaining the larval weight loss observed. Additionally, we fou…
Oxylipin mediated stress response of a miraculin-like protease inhibitor in Hexanoic acid primed eggplant plants infested by Colorado potato beetle
Insect-plant interactions are governed by a complex equilibrium between the mechanisms through which plant recognize insect attack and orchestrate downstream signaling events that trigger plant defense responses, and the mechanisms by which insects overcome plant defenses. Due to this tight and dynamic interplay, insight into the nature of the plant defense response can be gained by analyzing changes in the insect herbivores digestive system upon plant feeding. In this work we have identified a Solanum melongena miraculin-like protease inhibitor in the midgut juice of Colorado potato larvae feeding on eggplant plants treated with the natural inducer of plant defenses hexanoic acid. We analy…
Expression of miR159 Is Altered in Tomato Plants Undergoing Drought Stress.
In a scenario of global climate change, water scarcity is a major threat for agriculture, severely limiting crop yields. Therefore, alternatives are urgently needed for improving plant adaptation to drought stress. Among them, gene expression reprogramming by microRNAs (miRNAs) might offer a biotechnologically sound strategy. Drought-responsive miRNAs have been reported in many plant species, and some of them are known to participate in complex regulatory networks via their regulation of transcription factors involved in water stress signaling. We explored the role of miR159 in the response of Solanum lycopersicum Mill. plants to drought stress by analyzing the expression of sly-miR159 and …
Phenobarbital Induction of UDP-glucosyltransferase Activity in Drosophila melanogaster Meigen
The inducibility of UDP-glucosyltransferase activities towards the exogenous substrates 1-naphthol and 2-naphthol and the endogenous metabolite xanthurenic acid was demonstrated in Drosophila melanogaster Meigen larvae and adults using phenobarbital as an inducer. In adults, a 3.5-fold increase of glucosyltransferase activity toward xanthurenic acid and a 2.0-fold increase of the activity toward exogenous substrates (1-naphthol and 2-naphthol) was found. In larvae, maximum induction of all three UDP-glucosyltransferase activities (2.5-fold and 1.5-fold increase of the activity toward the exogenous and endogenous substrates, respectively) was achieved when insects, reared on solid medium, we…
Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to Bacillus thuringiensis Cry3Aa toxicity
Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highl…
Separation by FPLC chromatofocusing of UDP-glucosyltransferases from three developmental stages of Drosophila melanogaster.
Variation of UDP-glucosyltransferase activity, during Drosophila melanogaster development, was analyzed. The endogenous metabolite xanthurenic acid and the xenobiotic compounds 1-naphthol and 2-naphthol were used as substrates. Developmentally regulated differences were observed for the three substrates, suggesting the presence of UDP-glucosyltransferase isoenzymes. This was further confirmed by FPLC chromatofocusing on a Mono P column: seven peaks of UDP-glucosyltransferase activity (pHs: ≥6.3, 5.8, 5.5, 4.9, 4.5, 4.2, ≤4.0) with either single or overlapping substrate specificity were detected. A single xanthurenic acid:UDP-glucosyltransferase activity (pl 5.8) was found throughout develop…