Carmen Sara Hernández‐Rodríguez

Universitat de València

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Juan Ferré

Universitat de València

Jeroen Van Rie

Bio Base Europe

Baltasar Escriche

Universitat de València

Joel González‐Cabrera

Universitat de València

Patricia Hernández‐Martínez

Universitat de València

Primitivo Caballero

Universidad Publica de Navarra

Nicholas R. Waterfield

University of Warwick

Sara Moreno‐Martí

Universitat de València

ÍR

Íñigo Ruiz de Escudero

Universidad Publica de Navarra

Grace Taylor-Joyce

University of Warwick

Cooperative Institutions

Universitat de València

Instituto de Biomedicina de Valencia

Gobierno de Navarra

Agrobiotechnology Institute

National Center for Genetic Engineering and Biotechnology

Universidad Publica de Navarra

University of Warwick

Agricultural Research Service

Universidad de Navarra

United States Department of Agriculture

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B-Exotoxina de Bacillus thurigiensis: ¿bioinsecticida o metabolito no deseable?

Phytoma España: La revista profesional de sanidad vegetal (2002)

Juan Ferré Clara Martı́nez Manel Porcar Primitivo Caballero Carmen Sara Hernández‐Rodríguez

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Pdl1 Is a Putative Lipase that Enhances Photorhabdus Toxin Complex Secretion

PLoS Pathogens (2012)

Guowei Yang Carmen Sara Hernández‐Rodríguez Michael L. Beeton Paul A. Wilkinson Richard H. ffrench‐Constant

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4∶1∶1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in Gram negative pathogens.

Photorhabdus

Photorhabdus luminescens

Xenorhabdus

Virulence factor

Heterologous

Heterologous expression

10.1371/journal.ppat.1002692

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Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

PLoS ONE (2010)

Silvia Caccia Carmen Sara Hernández‐Rodríguez Rod Mahon Sharon Downes William James

Background Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. Methodology/Principal Findings Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. Conclusion/Significance This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.

Cry1Ac

Bacillus thuringiensis

Helicoverpa

Bt Cotton

10.1371/journal.pone.0009975

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Screening and identification ofvipgenes inBacillus thuringiensisstrains

Journal of Applied Microbiology (2009)

Carmen Sara Hernández‐Rodríguez Annemie Boets Jeroen Van Rie Juan Ferré

Aims: To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis. Methods and Results: Following a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48·9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains. Conclusions: Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study. Significance and Impact of the Study: This is the first time that vip1 and vip2 genes have been identified by PCR‐RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.

Bacillus thuringiensis

10.1111/j.1365-2672.2009.04199.x

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Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites

Applied and Environmental Microbiology (2009)

Janete Apparecida Desidério Carmen Sara Hernández‐Rodríguez Juan Ferré

ABSTRACT Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

Bacillus thuringiensis

10.1128/aem.02342-08

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Citations (151)

Insecticidal activity of Vip3Aa, Vip3Ad, Vip3Ae, and Vip3Af from Bacillus thuringiensis against lepidopteran corn pests

Journal of Invertebrate Pathology (2013)

Patricia Hernández‐Martínez Carmen Sara Hernández‐Rodríguez Jeroen Van Rie Baltasar Escriche Juan Ferré

Bacillus thuringiensis

Agrotis ipsilon

10.1016/j.jip.2013.02.001

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Citations (62)

Genomic structure and promoter analysis of pathogen‐induced repat genes from Spodoptera exigua

Insect Molecular Biology (2008)

Carmen Sara Hernández‐Rodríguez Juan Ferré Salvador Herrero

The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5'-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5'-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5'-untranslated region in the enhancement of gene expression.

Polyhedrin

Exigua

Autographa californica

10.1111/j.1365-2583.2008.00850.x

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Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

Applied and Environmental Microbiology (2008)

Carmen Sara Hernández‐Rodríguez Adri Van Vliet Nadine Bautsoens Jeroen Van Rie Juan Ferré

ABSTRACT For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125 I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera . Homologous-competition assays with 125 I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

Cry1Ac

Bacillus thuringiensis

10.1128/aem.01373-08

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Different binding sites for Bacillus thuringiensis Cry1Ba and Cry9Ca proteins in the European corn borer, Ostrinia nubilalis (Hübner)

Journal of Invertebrate Pathology (2014)

Patricia Hernández‐Martínez Carmen Sara Hernández‐Rodríguez Jeroen Van Rie Baltasar Escriche Juan Ferré

Ostrinia

European corn borer

Bacillus thuringiensis

10.1016/j.jip.2014.04.008

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thuringiensisProteins from Bacillus Vip3C, a Novel Class of Vegetative

Juan Ferré Primitivo Caballero Delia Muñoz Jeroen Van Rie Mireya Maeztu

(4, 19) which do not share se-quence homology with known Cry proteins and display insecti-cidal activity against a wide variety of lepidopterans (5, 21) andcoleopterans (19) and some sap-sucking insect pests (17). Themembers of the Vip3 family characterized to date exhibit activityagainst lepidopterans, and several of them do not compete withCryproteinsforbindingsites(12,18).Theyareclassiﬁedintotwosubfamilies(Vip3AandVip3B),andsomeareespeciallytoxicforspecieswithlittlesusceptibilitytoseveralCryproteins(4,14).AllofthesefeatureshavemadeVipsaresearchtargetforbroadeningthe host-range of

Homology

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