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Prof. Juan Ferre
ERI de Biotecnología y Biomedicina (BIOTECMED), Department of Genetics, Universitat de València, 46100 Burjassot, Spain

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Bacillus thuringiensis
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midgut
larvae
Cry1ac
Lepidoptera
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Communication
Published: 20 May 2021 in Toxins
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Laboratory selection for resistance of field populations is a well-known and useful tool to understand the potential of insect populations to evolve resistance to insecticides. It provides us with estimates of the frequency of resistance alleles and allows us to study the mechanisms by which insects developed resistance to shed light on the mode of action and optimize resistance management strategies. Here, a field population of Mythimna separata was subjected to laboratory selection with either Vip3Aa, Cry1Ab, or Cry1F insecticidal proteins from Bacillus thuringiensis. The population rapidly evolved resistance to Vip3Aa reaching, after eight generations, a level of >3061-fold resistance, compared with the unselected insects. In contrast, the same population did not respond to selection with Cry1Ab or Cry1F. The Vip3Aa resistant population did not show cross resistance to either Cry1Ab or Cry1F. Radiolabeled Vip3Aa was tested for binding to brush border membrane vesicles from larvae from the susceptible and resistant insects. The results did not show any qualitative or quantitative difference between both insect samples. Our data, along with previous results obtained with other Vip3Aa-resistant populations from other insect species, suggest that altered binding to midgut membrane receptors is not the main mechanism of resistance to Vip3Aa.

ACS Style

Yudong Quan; Jing Yang; Yueqin Wang; Patricia Hernández-Martínez; Juan Ferré; Kanglai He. The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors. Toxins 2021, 13, 364 .

AMA Style

Yudong Quan, Jing Yang, Yueqin Wang, Patricia Hernández-Martínez, Juan Ferré, Kanglai He. The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors. Toxins. 2021; 13 (5):364.

Chicago/Turabian Style

Yudong Quan; Jing Yang; Yueqin Wang; Patricia Hernández-Martínez; Juan Ferré; Kanglai He. 2021. "The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors." Toxins 13, no. 5: 364.

Review article
Published: 17 February 2021 in Microbiology and Molecular Biology Reviews
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Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives are bioinsecticides based on Bacillus thuringiensis .

ACS Style

Daniel Pinos; Ascensión Andrés-Garrido; Juan Ferré; Patricia Hernández-Martínez. Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins. Microbiology and Molecular Biology Reviews 2021, 85, 1 .

AMA Style

Daniel Pinos, Ascensión Andrés-Garrido, Juan Ferré, Patricia Hernández-Martínez. Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins. Microbiology and Molecular Biology Reviews. 2021; 85 (1):1.

Chicago/Turabian Style

Daniel Pinos; Ascensión Andrés-Garrido; Juan Ferré; Patricia Hernández-Martínez. 2021. "Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins." Microbiology and Molecular Biology Reviews 85, no. 1: 1.

Journal article
Published: 07 January 2021 in Annual Review of Entomology
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Insecticidal proteins from the bacterium Bacillus thuringiensis ( Bt) are used in sprayable formulations or produced in transgenic crops as the most successful alternatives to synthetic pesticides. The most relevant threat to sustainability of Bt insecticidal proteins (toxins) is the evolution of resistance in target pests. To date, high-level resistance to Bt sprays has been limited to one species in the field and another in commercial greenhouses. In contrast, there are currently seven lepidopteran and one coleopteran species that have evolved practical resistance to transgenic plants producing insecticidal Bt proteins. In this article, we present a review of the current knowledge on mechanisms of resistance to Bt toxins, with emphasis on key resistance genes and field-evolved resistance, to support improvement of Bt technology and its sustainability.

ACS Style

Juan Luis Jurat-Fuentes; David G. Heckel; Juan Ferré. Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis. Annual Review of Entomology 2021, 66, 121 -140.

AMA Style

Juan Luis Jurat-Fuentes, David G. Heckel, Juan Ferré. Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis. Annual Review of Entomology. 2021; 66 (1):121-140.

Chicago/Turabian Style

Juan Luis Jurat-Fuentes; David G. Heckel; Juan Ferré. 2021. "Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis." Annual Review of Entomology 66, no. 1: 121-140.

Editorial
Published: 10 December 2020 in Toxins
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Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date

ACS Style

Yolanda Bel; Juan Ferré; Patricia Hernández-Martínez. Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action. Toxins 2020, 12, 785 .

AMA Style

Yolanda Bel, Juan Ferré, Patricia Hernández-Martínez. Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action. Toxins. 2020; 12 (12):785.

Chicago/Turabian Style

Yolanda Bel; Juan Ferré; Patricia Hernández-Martínez. 2020. "Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action." Toxins 12, no. 12: 785.

Journal article
Published: 07 August 2020 in Nature Communications
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Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.

ACS Style

Rafael Núñez-Ramírez; Juanjo Huesa; Yolanda Bel; Juan Ferré; Patricia Casino; Ernesto Arias-Palomo. Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis. Nature Communications 2020, 11, 1 -9.

AMA Style

Rafael Núñez-Ramírez, Juanjo Huesa, Yolanda Bel, Juan Ferré, Patricia Casino, Ernesto Arias-Palomo. Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis. Nature Communications. 2020; 11 (1):1-9.

Chicago/Turabian Style

Rafael Núñez-Ramírez; Juanjo Huesa; Yolanda Bel; Juan Ferré; Patricia Casino; Ernesto Arias-Palomo. 2020. "Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis." Nature Communications 11, no. 1: 1-9.

Journal article
Published: 11 July 2020 in Journal of Invertebrate Pathology
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Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 protein by site-directed mutagenesis, distributed along the five structural domains of the protein. Ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). The results showed that, to render a wild type fragment pattern upon trypsin treatment, position 483 required an acidic residue, and position 552 an aromatic residue. Regarding toxicity, the change of Met34 to Lys34 significantly increased the toxicity of the protein for one of the three insect species tested (S. littoralis), whereas the other residue substitutions did not improve, or even decreased, insect toxicity, confirming their key role in the structure/function of the protein.

ACS Style

Núria Banyuls; Yudong Quan; Rosa María González-Martínez; Patricia Hernández-Martínez; Juan Ferré. Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis. Journal of Invertebrate Pathology 2020, 107439 .

AMA Style

Núria Banyuls, Yudong Quan, Rosa María González-Martínez, Patricia Hernández-Martínez, Juan Ferré. Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis. Journal of Invertebrate Pathology. 2020; ():107439.

Chicago/Turabian Style

Núria Banyuls; Yudong Quan; Rosa María González-Martínez; Patricia Hernández-Martínez; Juan Ferré. 2020. "Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis." Journal of Invertebrate Pathology , no. : 107439.

Article
Published: 10 July 2020 in Current Microbiology
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Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect’s resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore–crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of “functional mortality” was used to discriminate between “nontoxic” and “toxic” isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore–crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65–70 kDa. The LC–MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

ACS Style

Zahia Djenane; Maria Lázaro-Berenguer; Farida Nateche; Juan Ferré. Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria. Current Microbiology 2020, 77, 2904 -2914.

AMA Style

Zahia Djenane, Maria Lázaro-Berenguer, Farida Nateche, Juan Ferré. Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria. Current Microbiology. 2020; 77 (10):2904-2914.

Chicago/Turabian Style

Zahia Djenane; Maria Lázaro-Berenguer; Farida Nateche; Juan Ferré. 2020. "Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria." Current Microbiology 77, no. 10: 2904-2914.

Journal article
Published: 19 June 2020 in Toxins
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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 >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

ACS Style

Daniel Pinos; Maissa Chakroun; Anabel Millán-Leiva; Juan Luis Jurat-Fuentes; Denis J. Wright; Patricia Hernández-Martínez; Juan Ferré. Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony. Toxins 2020, 12, 409 .

AMA Style

Daniel Pinos, Maissa Chakroun, Anabel Millán-Leiva, Juan Luis Jurat-Fuentes, Denis J. Wright, Patricia Hernández-Martínez, Juan Ferré. Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony. Toxins. 2020; 12 (6):409.

Chicago/Turabian Style

Daniel Pinos; Maissa Chakroun; Anabel Millán-Leiva; Juan Luis Jurat-Fuentes; Denis J. Wright; Patricia Hernández-Martínez; Juan Ferré. 2020. "Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony." Toxins 12, no. 6: 409.

Preprint content
Published: 17 February 2020
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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, 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 of Heliothis 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 of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae show dramatically reduced levels of alkaline phosphatase activity, which was further confirmed by a strong down-regulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, its involvement as a receptor for the Vip3Aa protein was not supported by ligand blotting and viability assays with insect cells expressing HvmALP1. These data support that reduced alkaline phosphatase, previously observed in insect colonies resistant to Cry proteins from Bt, may also serve as an indirect marker that is not mechanistically involved in resistance to Vip3Aa.

ACS Style

Daniel Pinos; Maissa Chakroun; Anabel Millán-Leiva; Juan Luis Jurat-Fuentes; Denis J. Wright; Patricia Hernández-Martínez; Juan Ferré. Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens. 2020, 1 .

AMA Style

Daniel Pinos, Maissa Chakroun, Anabel Millán-Leiva, Juan Luis Jurat-Fuentes, Denis J. Wright, Patricia Hernández-Martínez, Juan Ferré. Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens. . 2020; ():1.

Chicago/Turabian Style

Daniel Pinos; Maissa Chakroun; Anabel Millán-Leiva; Juan Luis Jurat-Fuentes; Denis J. Wright; Patricia Hernández-Martínez; Juan Ferré. 2020. "Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens." , no. : 1.

Journal article
Published: 04 February 2020 in Toxins
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The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189–509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.

ACS Style

Joaquín Gomis-Cebolla; Rafael Ferreira dos Santos; Yueqin Wang; Javier Caballero; Primitivo Caballero; Kanglai He; Juan Jurat-Fuentes; Juan Ferré. Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests. Toxins 2020, 12, 99 .

AMA Style

Joaquín Gomis-Cebolla, Rafael Ferreira dos Santos, Yueqin Wang, Javier Caballero, Primitivo Caballero, Kanglai He, Juan Jurat-Fuentes, Juan Ferré. Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests. Toxins. 2020; 12 (2):99.

Chicago/Turabian Style

Joaquín Gomis-Cebolla; Rafael Ferreira dos Santos; Yueqin Wang; Javier Caballero; Primitivo Caballero; Kanglai He; Juan Jurat-Fuentes; Juan Ferré. 2020. "Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests." Toxins 12, no. 2: 99.

Journal article
Published: 21 June 2019 in Toxins
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Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of the trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning of Vip3Af, we selected mutants with an altered proteolytic pattern. Based on protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains which match some of the domains proposed independently by two in silico models. Domain I ranges amino acids (aa) 12–198, domain II aa199–313, domain III aa314–526, domain IV aa527–668, and domain V aa669–788. The effect of some mutations on the ability to form a tetrameric molecule revealed that domains I–II are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and destroy the tetrameric form upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to the Vip3 family of proteins.

ACS Style

Yudong Quan; Juan Ferré; Quan. Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants. Toxins 2019, 11, 368 .

AMA Style

Yudong Quan, Juan Ferré, Quan. Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants. Toxins. 2019; 11 (6):368.

Chicago/Turabian Style

Yudong Quan; Juan Ferré; Quan. 2019. "Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants." Toxins 11, no. 6: 368.

Journal article
Published: 22 March 2019 in Toxins
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ABC proteins are primary-active transporters that require the binding and hydrolysis of ATP to transport substrates across the membrane. Since the first report of an ABCC2 transporter as receptor of Cry1A toxins, the number of ABC transporters known to be involved in the mode of action of Cry toxins has increased. In Spodoptera exigua, a mutation in the SeABCC2 gene is described as genetically linked to resistance to the Bt-product XentariTM. This mutation affects an intracellular domain involved in ATP binding, but not the extracellular loops. We analyzed whether this mutation affects the role of the SeABCC2 as a functional receptor to Cry1A toxins. The results show that Sf21 cells expressing the truncated form of the transporter were susceptible to Cry1A toxins. Moreover, specific Cry1Ac binding was observed in those cells expressing the truncated SeABCC2. Additionally, no differences in the irreversible Cry1Ac binding component (associated with the toxin insertion into the membrane) were observed when tested in Sf21 cells expressing either the full-length or the truncated form of the SeABCC2 transporter. Therefore, our results point out that the partial lack of the nucleotide binding domain II in the truncated transporter does not affect its functionality as a Cry1A receptor.

ACS Style

Daniel Pinos; María Martínez-Solís; Salvador Herrero; Juan Ferré; Patricia Hernández-Martínez. The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II. Toxins 2019, 11, 172 .

AMA Style

Daniel Pinos, María Martínez-Solís, Salvador Herrero, Juan Ferré, Patricia Hernández-Martínez. The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II. Toxins. 2019; 11 (3):172.

Chicago/Turabian Style

Daniel Pinos; María Martínez-Solís; Salvador Herrero; Juan Ferré; Patricia Hernández-Martínez. 2019. "The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II." Toxins 11, no. 3: 172.

Journal article
Published: 04 November 2018 in Toxins
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The oriental armyworm (OAW), Mythimna separata (Walker), is a destructive pest of agricultural crops in Asia and Australia. Commercialized Bt crops have performed very well against their target pests; however, very few studies have been done on the susceptibility of OAW to Bt toxins in either sprays or expressed in Bt crops. In this work, we evaluated the toxicities of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, Cry2Aa, Cry2Ab, Cry1Ie, Vip3Aa19, Vip3Aa16, and Vip3Ca against OAW neonate larvae, as well as the interaction between Cry and Vip toxins. The results from bioassays revealed that LC50 (lethal concentration for 50% mortality) values ranged from 1.6 to 78.6 μg/g (toxin/diet) for those toxins. Among them, Vip3 proteins, along with Cry1A proteins and Cry2Aa, were the ones with the highest potency, with LC50 values ranging from 1.6 to 7.4 μg/g. Synergism between Cry and Vip toxins was observed, being high in the combination of Vip3Aa16 with Cry1 toxins, with synergetic factors ranging from 2.2 to 9.2. The Vip3Ca toxin did not show any synergistic effect with any of the toxins tested. These results can help in designing new combinations of pyramiding genes in Bt crops, as well as in recombinant bacteria, for the control of OAW as well as for resistance management programs.

ACS Style

Jing Yang; Yudong Quan; Prabu Sivaprasath; Muhammad Zeeshan Shabbir; Zhenying Wang; Juan Ferré; Kanglai He. Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker). Toxins 2018, 10, 454 .

AMA Style

Jing Yang, Yudong Quan, Prabu Sivaprasath, Muhammad Zeeshan Shabbir, Zhenying Wang, Juan Ferré, Kanglai He. Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker). Toxins. 2018; 10 (11):454.

Chicago/Turabian Style

Jing Yang; Yudong Quan; Prabu Sivaprasath; Muhammad Zeeshan Shabbir; Zhenying Wang; Juan Ferré; Kanglai He. 2018. "Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker)." Toxins 10, no. 11: 454.

Research article
Published: 01 November 2018 in PLOS ONE
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Bacillus thuringiensis (Bt) constitutes the active ingredient of many successful bioinsecticides used in agriculture. In the present study, the genetic diversity and toxicity of Bt isolates was investigated by characterization of native isolates originating from soil, fig leaves and fruits from a Turkish collection. Among a total of 80 Bt isolates, 18 of them were found carrying a vip3 gene (in 23% of total), which were further selected. Insecticidal activity of spore/crystal mixtures and their supernatants showed that some of the Bt isolates had significantly more toxicity against some lepidopteran species than the HD1 reference strain. Five isolates were analyzed by LC-MS/MS to determine the Cry protein composition of their crystals. The results identified the Cry1Ac protein and a Cry2A-type protein in all isolates, Cry1Ea in 3 of them and Cry1Aa in one. The sequence analysis of the new vip3 genes showed that they had a high similarity to either vip3Aa, vip3Af or vip3Ag (94–100%). The vip3Aa gene of the 6A Bt isolate was cloned and sequenced. The protein was named Vip3Aa65 by the Bacillus thuringiensis Nomenclature Committee. The expressed and purified Vip3Aa65 protein was tested against five lepidopteran species and its toxicity compared to that of a reference protein (Vip3Aa16). Both proteins had similar toxicity against Grapholita molesta and Helicoverpa armigera, whereas Vip3Aa65 was less active than Vip3Aa16 against three species from the Spodoptera genus. A tetrameric structure of the Vip3Aa65 protein was detected by gel filtration chromatography. The study revealed some isolates with high insecticidal activity which can be considered promising candidates to be used in pest control.

ACS Style

Burcu Şahin; Joaquín Gomis-Cebolla; Hatice Güneş; Juan Ferré. Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins. PLOS ONE 2018, 13, e0206813 .

AMA Style

Burcu Şahin, Joaquín Gomis-Cebolla, Hatice Güneş, Juan Ferré. Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins. PLOS ONE. 2018; 13 (11):e0206813.

Chicago/Turabian Style

Burcu Şahin; Joaquín Gomis-Cebolla; Hatice Güneş; Juan Ferré. 2018. "Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins." PLOS ONE 13, no. 11: e0206813.

Journal article
Published: 01 November 2018 in Scientific Reports
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A 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 with Vip3A, and does not share sites with Cry1F or Cry2A. In agreement with the resulting binding site model, Vip3A-resistant insects were also cross-resistant to the modified Vip3C protein. Furthermore, maize plants expressing the modified Vip3C protein, but not those expressing Cry1F protein, were protected against Cry1F-resistant S. frugiperda in field trials.

ACS Style

Theodore W. Kahn; Maissa Chakroun; Jayme Williams; Tom Walsh; Bill James; Jessica Monserrate; Juan Ferré. Efficacy and Resistance Management Potential of a Modified Vip3C Protein for Control of Spodoptera frugiperda in Maize. Scientific Reports 2018, 8, 16204 .

AMA Style

Theodore W. Kahn, Maissa Chakroun, Jayme Williams, Tom Walsh, Bill James, Jessica Monserrate, Juan Ferré. Efficacy and Resistance Management Potential of a Modified Vip3C Protein for Control of Spodoptera frugiperda in Maize. Scientific Reports. 2018; 8 (1):16204.

Chicago/Turabian Style

Theodore W. Kahn; Maissa Chakroun; Jayme Williams; Tom Walsh; Bill James; Jessica Monserrate; Juan Ferré. 2018. "Efficacy and Resistance Management Potential of a Modified Vip3C Protein for Control of Spodoptera frugiperda in Maize." Scientific Reports 8, no. 1: 16204.

Journal article
Published: 16 August 2018 in International Journal of Biological Macromolecules
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Vip3 proteins are secretable proteins from Bacillus thuringiensis with important characteristics for the microbiological control of agricultural pests. The exact details of their mode of action are yet to be disclosed and the crystallographic structure is still unknown. Vip3 proteins are expressed as protoxins that have to be activated by the insect gut proteases. A previous study on the peptidase processing of Vip3Aa revealed that the protoxin produced artefactual band patterns by SDS-PAGE due to the differential stability of this protein and the peptidases to SDS and heating (Bel et al., 2017 Toxins 9:131). To determine whether this phenomenon also applies to other Vip3A proteins, here we chose a different Vip3A protein (Vip3Af) and subjected it to commercial trypsin and midgut juice from a target insect species (Spodoptera frugiperda). The misleading degradation patterns were also observed with Vip3Af, both with trypsin and midgut juice. However, gel filtration chromatography showed that, under native conditions, Vip3Af is found as a tetramer and that peptidases only act upon primary cleavage sites. The proteolytic cleavage renders two fragments of approximately 20 kDa and 65 kDa which remain together in the tretameric structure and that are no further processed even at high peptidase concentrations.

ACS Style

Núria Banyuls; Patricia Hernández-Martínez; Yudong Quan; Juan Ferré. Artefactual band patterns by SDS-PAGE of the Vip3Af protein in the presence of proteases mask the extremely high stability of this protein. International Journal of Biological Macromolecules 2018, 120, 59 -65.

AMA Style

Núria Banyuls, Patricia Hernández-Martínez, Yudong Quan, Juan Ferré. Artefactual band patterns by SDS-PAGE of the Vip3Af protein in the presence of proteases mask the extremely high stability of this protein. International Journal of Biological Macromolecules. 2018; 120 ():59-65.

Chicago/Turabian Style

Núria Banyuls; Patricia Hernández-Martínez; Yudong Quan; Juan Ferré. 2018. "Artefactual band patterns by SDS-PAGE of the Vip3Af protein in the presence of proteases mask the extremely high stability of this protein." International Journal of Biological Macromolecules 120, no. : 59-65.

Journal article
Published: 01 June 2018 in Journal of Invertebrate Pathology
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Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect colonies from different species to determine whether resistance to other insecticidal proteins confers cross-resistance to Vip3 proteins. As expected, the colonies resistant to Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) were not cross-resistant to Vip3 proteins. In contrast, H. armigera colonies resistant to Vip3Aa or Vip3Aa/Cry2Ab showed cross-resistance to the Vip3Ca protein. Moreover, the Vip3Ca protein was highly toxic to O. furnacalis (LC50 not significantly different from that of Cry1Ab), whereas the Vip3Aa protein only showed moderate growth inhibition at the highest concentration tested (100 µg/g of diet). These results extend the cross-resistance studies between Vip3 and Cry proteins, show for the first time cross-resistance between proteins within the Vip3 subfamily, and points to O. furnacalis as a target for the Vip3Ca protein.

ACS Style

Joaquin Gomis-Cebolla; Yuequin Wang; Yudong Quan; Kanglai He; Tom Walsh; Bill James; Sharon Downes; Wendy Kain; Ping Wang; Kathy Leonard; Tom Morgan; Brenda Oppert; Juan Ferré. Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins. Journal of Invertebrate Pathology 2018, 155, 64 -70.

AMA Style

Joaquin Gomis-Cebolla, Yuequin Wang, Yudong Quan, Kanglai He, Tom Walsh, Bill James, Sharon Downes, Wendy Kain, Ping Wang, Kathy Leonard, Tom Morgan, Brenda Oppert, Juan Ferré. Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins. Journal of Invertebrate Pathology. 2018; 155 ():64-70.

Chicago/Turabian Style

Joaquin Gomis-Cebolla; Yuequin Wang; Yudong Quan; Kanglai He; Tom Walsh; Bill James; Sharon Downes; Wendy Kain; Ping Wang; Kathy Leonard; Tom Morgan; Brenda Oppert; Juan Ferré. 2018. "Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins." Journal of Invertebrate Pathology 155, no. : 64-70.

Journal article
Published: 15 May 2018 in Scientific Reports
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Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167–272 and amino acids 689–741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.

ACS Style

N. Banyuls; C. S. Hernández-Rodríguez; J. Van Rie; J. Ferré. Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects. Scientific Reports 2018, 8, 7539 .

AMA Style

N. Banyuls, C. S. Hernández-Rodríguez, J. Van Rie, J. Ferré. Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects. Scientific Reports. 2018; 8 (1):7539.

Chicago/Turabian Style

N. Banyuls; C. S. Hernández-Rodríguez; J. Van Rie; J. Ferré. 2018. "Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects." Scientific Reports 8, no. 1: 7539.

Journal article
Published: 10 May 2018 in Toxins
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The combined analysis of genomic and proteomic data allowed us to determine which cry and vip genes are present in a Bacillus thuringiensis (Bt) isolate and which ones are being expressed. Nine Bt isolates were selected from Spanish collections of Bt based on their vip1 and vip2 gene content. As a first step, nine isolates were analyzed by PCR to select those Bt isolates that contained genes with the lowest similarity to already described vip1 and vip2 genes (isolates E-SE10.2 and O-V84.2). Two selected isolates were subjected to a combined genomic and proteomic analysis. The results showed that the Bt isolate E-SE10.2 codifies for two new vegetative proteins, Vip2Ac-like_1 and Sip1Aa-like_1, that do not show expression differences at 24 h vs. 48 h and are expressed in a low amount. The Bt isolate O-V84.2 codifies for three new vegetative proteins, Vip4Aa-like_1, Vip4Aa-like_2, and Vip2Ac-like_2, that are marginally expressed. The Vip4Aa-like_1 protein was two-fold more abundant at 24 h vs. 48 h, while the Vip4Aa-like_2 was detected only at 24 h. For Vip2Ac-like_2, no differences in expression were found at 24 h vs. 48 h. Moreover, the parasporal crystal of the E-SE10.2 isolate contains a single type of crystal protein, Cry23Aa-like, while the parasporal crystal from O-V84.2 contains three kinds of crystal proteins: 7.0–9.8% weight of Cry45Aa-like proteins, 35–37% weight of Cry32-like proteins and 2.8–4.3% weight of Cry73-like protein.

ACS Style

Joaquín Gomis-Cebolla; Ana Paula Scaramal Ricietto; Juan Ferré. A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal. Toxins 2018, 10, 193 .

AMA Style

Joaquín Gomis-Cebolla, Ana Paula Scaramal Ricietto, Juan Ferré. A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal. Toxins. 2018; 10 (5):193.

Chicago/Turabian Style

Joaquín Gomis-Cebolla; Ana Paula Scaramal Ricietto; Juan Ferré. 2018. "A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal." Toxins 10, no. 5: 193.

Editorial
Published: 09 December 2017 in Toxins
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Ferré, J.; Escriche, B. Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture. Toxins 2017, 9, 396. Ferré J, Escriche B. Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture. Toxins. 2017; 9(12):396. Ferré, Juan; Escriche, Baltasar. 2017. "Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture." Toxins 9, no. 12: 396.

ACS Style

Juan Ferré; Baltasar Escriche. Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture. Toxins 2017, 9, 396 .

AMA Style

Juan Ferré, Baltasar Escriche. Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture. Toxins. 2017; 9 (12):396.

Chicago/Turabian Style

Juan Ferré; Baltasar Escriche. 2017. "Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture." Toxins 9, no. 12: 396.