0000000000023531
AUTHOR
Patricia Hernández-martínez
showing 26 related works from this author
Hetero-oligomerization of Bacillus thuringiensis Cry1A proteins enhance binding to the ABCC2 transporter of Spodoptera exigua
2021
The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. …
Different binding sites for Bacillus thuringiensis Cry1Ba and Cry9Ca proteins in the European corn borer, Ostrinia nubilalis (Hübner).
2014
Binding studies using (125)I-Cry9Ca and biotinylated-Cry1Ba proteins showed the occurrence of independent binding sites for these proteins in Ostrinia nubilalis. Our results, along with previously available binding data, indicate that combinations of Cry1A or Cry1Fa proteins with Cry1Ba and/or Cry9Ca could be a good strategy for the resistance management of O. nubilalis.
Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultu…
2021
Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete f…
A new gene superfamily of pathogen-response (repat) genes in Lepidoptera: Classification and expression analysis
2012
Repat (REsponse to PAThogens) genes were first identified in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae) in response to Bacillus thuringiensis and baculovirus exposure. Since then, additional repat gene homologs have been identified in different studies. In this study the comprehensive larval transcriptome from S. exigua was analyzed for the presence of novel repat-homolog sequences. These analyses revealed the presence of at least 46 repat genes in S. exigua, establishing a new gene superfamily in this species. Phylogenetic analysis and studies of conserved motifs in these hypothetical proteins have allowed their classification in two main classes, αREPAT and βREPAT. Studies o…
Artefactual band patterns by SDS-PAGE of the Vip3Af protein in the presence of proteases mask the extremely high stability of this protein.
2018
Abstract 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…
Reduced membrane-bound alkaline phosphatase does not affect binding of Vip3Aa in a Heliothis virescens resistant colony
2020
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 >
Binding analysis of Bacillus thuringiensis Cry1 proteins in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae).
2015
Sugarcane borer (Diatraea saccharalis, F.) is an important corn pest in South America and United States. The aim of the present study was to analyze the susceptibility and binding interactions of three Cry1A proteins and Cry1Fa in a Brazilian D. saccharalis population. The results showed that Cry1Ab was the most active, followed by Cry1Ac, Cry1Fa and Cry1Aa. All Cry1-biotinylated proteins tested bound specifically to the D. saccharalis brush border membrane vesicles (BBMV). Heterologous competition assays showed shared binding sites for all Cry1A proteins and another one shared by Cry1Fa and Cry1Ab. Thus, pyramiding Cry1Aa/Cry1Ac and Cry1F proteins would be a recommended strategy for managi…
Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis
2007
Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC(50) values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and …
Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens
2020
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…
Susceptibility, mechanisms of response and resistance to Bacillus thuringiensis toxins in Spodoptera spp.
2016
Bioinsecticides based on Bacillus thuringiensis have long been used as an alternative to synthetic insecticides to control insect pests. In this review, we focus on insects of the genus Spodoptera, including relevant polyphagous species that are primary and secondary pests of many crops, and how B. thuringiensis toxins can be used for Spodoptera spp. pest management. We summarize the main findings related to susceptibility, midgut binding specificity, mechanisms of response and resistance of this insect genus to B. thuringiensis toxins.
Vip3C, a novel class of vegetative insectidal proteins from Bacillus thuringiensis
2012
Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 οg/cm 2. © 2012, American Society for Microbiology.
Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.
2016
The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90 kDa) was processed to an approximately 70 kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately…
The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors
2021
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…
Insecticidal activity of Vip3Aa, Vip3Ad, Vip3Ae, and Vip3Af from Bacillus thuringiensis against lepidopteran corn pests.
2012
Vip3Aa, Vip3Ad, Vip3Ae, and Vip3Af proteins from Bacillus thuringiensis were tested for their toxicity against Spodoptera frugiperda and Agrotis ipsilon. Vip3Ad was non-toxic to the two species. Vip3Ae and Vip3Af were significantly more toxic than Vip3Aa against S. frugiperda, both as protoxins and as toxins. Against A. ipsilon, Vip3Ae protoxin was more toxic than Vip3Aa and Vip3Af protoxins. Purification by metal-chelate affinity chromatography significantly affected Vip3Ae toxicity against the two insect species.
Specific binding of radiolabeled Cry1Fa insecticidal protein from Bacillus thuringiensis to midgut sites in lepidopteran species
2012
ABSTRACT Cry1Fa insecticidal protein was successfully radiolabeled with 125 I-Na. Specific binding to brush border membrane vesicles was shown for the lepidopteran species Ostrinia nubilalis , Spodoptera frugiperda , Spodoptera exigua , Helicoverpa armigera , Heliothis virescens , and Plutella xylostella . Homologous competition assays were performed to obtain equilibrium binding parameters ( K d [dissociation constant] and R t [concentration of binding sites]) for these six insect species.
Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrin…
2013
First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the c…
Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentida…
2014
ABSTRACT Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas , which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-…
Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis
2021
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 …
Lack of Cry1Fa binding to the midgut brush border membrane in a resistant colony of Plutella xylostella moths with a mutaton in the ABCC2 locus
2012
ABSTRACT Previous studies reported “mode 1” Bacillus thuringiensis resistance in a colony of diamondback moths (NO-QA), and recently, this resistance has been mapped to an ABC transporter ( ABCC2 ) locus. We report the lack of binding of Cry1Fa to insects derived from this colony and compare our data with those from other insects with ABCC2 -associated resistance.
Role of Bacillus thuringiensis Cry1A toxins domains in the binding to the ABCC2 receptor from Spodoptera exigua
2018
Abstract Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf2…
Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua
2010
Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari (TM), a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression …
The Spodoptera exigua ABCC2 Acts as a Cry1A Receptor Independently of its Nucleotide Binding Domain II
2019
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 express…
The Independent Biological Activity of Bacillus thuringiensis Cry23Aa Protein Against Cylas puncticollis
2020
The Cry23Aa/Cry37Aa proteins from Bacillus thuringiensis (Bt) have been described toxic to Cylas puncticollis larvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity toward C. puncticollis larvae has been assessed. The results showed that both proteins were toxic for C. puncticollis larvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity against C. puncticollis larvae. Also, the bin…
Broad-spectrum cross-resistance in Spodoptera exigua from selection with a marginally toxic Cry protein.
2009
BACKGROUND:Spodoptera exigua (Hubner) has developed resistance to a wide range of chemical insecticides. Products based on Bacillus thuringiensis Cry toxins are used in integrated pest management as an ecologically friendly alternative for pest control. Since there are few B. thuringiensis Cry proteins highly active against S. exigua, it is desirable to apply appropriate resistance management strategies to prevent the evolution of resistance to these proteins. RESULTS:Spodoptera exigua larvae were selected with Cry1Ab, a protein with low activity against this pest. Selected larvae developed > 30-fold resistance to Cry1Ab in 13 generations, relative to an unselected strain. The estimated rea…
Increase in midgut microbiota load induces an apparent immune priming and increases tolerance to Bacillus thuringiensis
2010
Summary The insect immune system is comprised of both humoral and cellular components that are mobilized in response to parasitic or pathogenic infections. Activation of the immune response implies a consid- erable expenditure of energy and that is why insects rely on inducible pathways that are activated after coming into contact with the pathogenic agent. Known as immune priming, insects can prolong the activation of the immune response and transmit their immune status to the next generation. Starting from a laboratory colony of the lepidopteran Spodoptera exigua and using the lytic zone assay as a measure of the immune status, we selected for a sub-colony with high levels of immune activ…
Unshared binding sites for Bacillus thuringiensis Cry3Aa and Cry3Ca proteins in the weevil Cylas puncticollis (Brentidae)
2016
Bacillus thuringiensis Cry3Aa and Cry3Ca proteins have been reported to be toxic against the African sweetpotato pest Cylas puncticollis. In the present work, the binding sites of these proteins in C. puncticollis brush border vesicles suggest the occurrence of different binding sites, but only one of them is shared. Our results suggest that pest resistance mediated by alteration of the shared Cry-receptor binding site might not render both Cry proteins ineffective.