0000000000160344
AUTHOR
Maissa Chakroun
Biochemical and molecular study of the Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) mode of action in Spodoptera species
Las proteínas insecticidas vegetativas (Vip) constituyen una nueva familia de toxinas producidas durante la fase de crecimiento vegetativo de diferentes cepas de Bacillus y principalmente por Bacillus thuringiensis (Bt). Esta familia de proteínas está representada por 4 miembros: Vip1, Vip2, Vip3 y la recientemente descrita Vip4. Las toxinas binarias Vip1 y Vip2 son activas contra coleópteros y homópteros; las proteínas Vip3 son activas contra lepidópteros, sin embargo, los insectos diana para la proteína Vip4 no se conocen todavía. La determinación del modo de acción de Vip1 y Vip2 fue fácil ya que mostraron una homología de secuencia significativa con las toxinas clostridiales C2 y C3, re…
Reduced membrane-bound alkaline phosphatase does not affect binding of Vip3Aa in a Heliothis virescens resistant colony
The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >
Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis
Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed t…
Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens
ABSTRACTThe Vip3Aa insecticidal protein fromBacillus thuringiensis(Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests, thus understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, a laboratory-selected colony ofHeliothis virescens(Vip-Sel) highly resistant to the Vip3Aa protein was used to test whether an alteration of membrane receptors in the insect midgut might explain the resistance phenotype. Binding of125I-labeled Vip3Aa to brush border membran…
Overproduction of the Bacillus thuringiensis Vip3Aa16 toxin and study of its insecticidal activity against the carob moth Ectomyelois ceratoniae
Abstract The vip3Aa16 gene of Bacillus thuringiensis strain BUPM95 was cloned and expressed in Escherichia coli . Optimization of Vip3A16 protein expression was conducted using Plackett–Burman design and response surface methodology. Accordingly, the optimum Vip3A16 toxin production was 170 μg/ml at 18 h post-induction time and 39 °C post-induction temperature. This corresponds to an improvement of 21 times compared to the starting conditions. The insecticidal activity, evaluated against Ectomyelois ceratoniae , displayed an LC 50 value of 40 ng/cm 2 and the midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formati…
Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein
The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae rem…
Characterization of the resistance to Vip3Aa in Helicoverpa armigera from Australia and the role of midgut processing and receptor binding.
AbstractCrops expressing genes from Bacillus thuringiensis (Bt crops) are among the most successful technologies developed for the control of pests but the evolution of resistance to them remains a challenge. Insect resistant cotton and maize expressing the Bt Vip3Aa protein were recently commercialized, though not yet in Australia. We found that, although relatively high, the frequency of alleles for resistance to Vip3Aa in field populations of H. armigera in Australia did not increase over the past four seasons until 2014/15. Three new isofemale lines were determined to be allelic with previously isolated lines, suggesting that they belong to one common gene and this mechanism is relative…
Ephestia kuehniella tolerance to Bacillus thuringiensis Cry1Aa is associated with reduced oligomer formation
The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endotoxins from Bacillus thuringiensis kurstaki BNS3 was studied. Both toxins bound specifically to the BBMV of E. kuehniella. The result of the ligand blot showed that Cry1Ac bound to three putative receptors of about 100, 65 and 80 kDa and Cry1Aa interacted only with a 100 kDa protein. Pronase digestion of the BBMV-bound toxins was used to analyze the toxin insertion. Both toxins inserted into the BBMV as monomers however, a 14 kDa peptide of α4-α5 which correspond to the oligomeric form of this peptide was detected in case of Cry1Ac only. Analysis of the in vitro oligomerisation of these toxins in…
Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species
Abstract Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera speci…
Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin
BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22kDa, whereas the activation of Vip3Aa16 by S. littoralis …
Correction for Chakroun et al., Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria
Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no …
In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125 I Radiolabeling
ABSTRACT Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the…
Isolating, characterising and identifying a Cry1Ac resistance mutation in field populations of Helicoverpa punctigera
AbstractTransgenic cotton expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been grown in Australia for over 20 years and resistance remains the biggest threat. The native moth, Helicoverpa punctigera is a significant pest of cotton. A genotype causing resistance to Cry1Ac in H. punctigera was isolated from the field and a homozygous line established. The phenotype is recessive and homozygous individuals possess 113 fold resistance to Cry1Ac. Individuals that carry Cry1Ac resistance genes are rare in Australia with a frequency of 0.033 being detected in field populations. RNAseq, RT-PCR and DNA sequencing reveals a single nucleotide polymorphism at a splice site in the c…
Efficacy and Resistance Management Potential of a Modified Vip3C Protein for Control of Spodoptera frugiperda in Maize
AbstractA modified Vip3C protein has been developed that has a spectrum of activity that has the potential to be commercially useful for pest control, and shows good efficacy against Spodoptera frugiperda in insect bioassays and field trials. For the first time Vip3A and Vip3C proteins have been compared to Cry1 and Cry2 proteins in a complete set of experiments from insect bioassays to competition binding assays to field trials, and the results of these complementary experiments are in agreement with each other. Binding assays with radiolabelled toxins and brush border membrane vesicles from S. frugiperda and Helicoverpa armigera show that the modified Vip3C protein shares binding sites wi…
Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria
SUMMARY Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3…
ABCC transporters mediate insect resistance to multiple Bt toxins revealed by bulk segregant analysis
[EN] Background: Relatively recent evidence indicates that ABCC2 transporters play a main role in the mode of action of Bacillus thuringiensis (Bt) Cry1A-type proteins. Mapping of major Cry1A resistance genes has linked resistance to the ABCC2 locus in Heliothis virescens, Plutella xylostella, Trichoplusia ni and Bombyx mori, and mutations in this gene have been found in three of these Bt-resistant strains. Results: We have used a colony of Spodoptera exigua (Xen-R) highly resistant to a Bt commercial bioinsecticide to identify regions in the S. exigua genome containing loci for major resistance genes by using bulk segregant analysis (BSA). Results reveal a region containing three genes fro…