Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.399
Filtrar
1.
J Agric Food Chem ; 69(40): 11816-11824, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34596393

RESUMO

The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Larva/genética , Mariposas/genética
2.
Braz J Biol ; 83: e246436, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495159

RESUMO

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.


Assuntos
Proteínas Hemolisinas , Mariposas , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Fertilizantes , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva , Nitrogênio , Fósforo , Plantas Geneticamente Modificadas/genética
3.
Neotrop Entomol ; 50(5): 804-811, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34398398

RESUMO

Bacillus thuringiensis (Bt)-based bioinsecticides and transgenic plants expressing proteins with insecticidal activity (Cry and Vip) have been successfully used in several integrated pest management programs worldwide. Lepidoptera comprise some of the most economically important insect pests of the major agricultural crops. In this study, the toxicity of 150 Bt strains was evaluated against Helicoverpa armigera (Hübner) larvae. Eight strains (426, 520B, 1636, 1641, 1644, 1648, 1657 and 1658) showed high insecticide activity against H. armigera and were therefore tested against Anticarsia gemmatalis (Hübner), Spodoptera cosmioides (Walker), Chrysodeixis includens (Walker), and Diatraea saccharalis (Fabricius) larvae. Our results showed that most of the Bt strains were also toxic to these lepidopteran species. The biochemical and molecular analyses of these strains revealed that they had a similar protein profile; however, their cry and vip gene contents were variable. In addition, the median lethal concentration (LC50) of the selected strains indicated that the strains 1636, 1641, and 1658 were the most effective against H. armigera, showing LC50 values of 185.02, 159.44, and 192.98 ng/cm2, respectively. Our results suggest that the selected Bt strains have great potential to control the lepidopteran pests H. armigera, A. gemmatalis, D. saccharalis, S. cosmioides, and C. includes.


Assuntos
Bacillus thuringiensis , Agentes de Controle Biológico , Mariposas , Controle Biológico de Vetores , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas/toxicidade , Proteínas Hemolisinas , Larva/microbiologia , Mariposas/microbiologia
4.
Acta Trop ; 223: 106088, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34389325

RESUMO

Some Bacillus thuringiensis (Bt) strains produce dipteran-active toxins and can control larval mosquitoes. We identified a novel mosquitocidal toxin named Xpp81Aa1 with the thioredoxin domain from Bt strain HSY204. This toxin has very little sequence similarity to the three-domain Cry toxin and Cyt toxins and has significant toxicity to Aedes aegypti larvae. A safety assessment indicated that the Xpp81Aa1 toxin has no cytocidal activity against red blood cells and did not induce allergic reactions. The Xpp81Aa1 toxin exhibited a synergistic effect in combination with Cry2Aa and Cry4Aa protein toxins. Thus, the Xpp81Aa1 toxin could be a good candidate for mosquito control applications to reduce the mosquito-borne disease.


Assuntos
Aedes , Toxinas de Bacillus thuringiensis , Controle de Mosquitos , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Larva
5.
GM Crops Food ; 12(1): 382-395, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34193022

RESUMO

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Resistência a Inseticidas , Larva , Metanol , Plantas Geneticamente Modificadas
6.
J Agric Food Chem ; 69(36): 10407-10418, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34319733

RESUMO

Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.


Assuntos
Bacillus thuringiensis , Técnicas Biossensoriais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Imunoensaio
7.
Arch Insect Biochem Physiol ; 108(2): e21834, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34288075

RESUMO

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Caderinas/metabolismo , Besouros/efeitos dos fármacos , Besouros/metabolismo , Besouros/microbiologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/microbiologia , Controle Biológico de Vetores , Interferência de RNA
8.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198929

RESUMO

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.


Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Receptores de Superfície Celular/genética , Animais , Bacillus thuringiensis/genética , Endotoxinas/genética , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Mutação/genética , Regiões Promotoras Genéticas/genética
9.
Pest Manag Sci ; 77(10): 4593-4606, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34092019

RESUMO

BACKGROUND: Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS: Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION: This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Larva/metabolismo , Mariposas/metabolismo , Ligação Proteica
10.
Insect Biochem Mol Biol ; 135: 103608, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34119653

RESUMO

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that kill insect pests. Insect specificity of Cry toxins relies on their binding to larval gut membrane proteins such as cadherin and ATP-binding cassette (ABC) transporter proteins. Mutations in ABC transporters have been implicated in high levels of resistance to Cry toxins in multiple pests. Spodoptera frugiperda is an insect pest susceptible to Cry1Fa and Cry1Ab toxins while Mythimna separata is tolerant to Cry1Fa and less susceptible to Cry1Ab. Here, we analyzed the potential role of ABCC2 in determining the susceptibility of S. frugiperda to Cry1Fa and Cry1Ab, by expressing SfABCC2 or MsABCC2 in Hi5 insect cell line and by the systematic replacements of extracellular loops (ECLs) between these two proteins. Expression of SfABCC2 in Hi5 conferred susceptibility to both Cry1Fa and Cry1Ab, in contrast to the expression of MsABCC2 that mediated low toxicity to Cry1Ab and no toxicity to Cry1Fa in agreement with their larvicidal toxicities. The SfABCC2 and MsABCC2 amino acid sequences showed differential residues among ECL1, ECL2, ECL4 and ECL6 loops, while ECL3 and ECL5 share the same primary sequence. The exchange of ECLs between SfABCC2 and MsABCC2 demonstrated that ECL4 and ECL2 contribute to Cry1Fa toxicity, where ECL4 plays a major role. The medium region (named M2) of ECL4 was identified as the most important region of SfABCC2 involved in Cry1Fa toxicity as shown by point mutations in this region. These findings will be helpful to understand the mechanisms of action of Bt toxins in S. frugiperda.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Resistência a Inseticidas/genética , Mariposas , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Animais , Proteínas de Bactérias/farmacologia , Linhagem Celular , Endotoxinas/farmacologia , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Spodoptera/metabolismo
11.
Nat Commun ; 12(1): 3380, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099714

RESUMO

Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Produtos Agrícolas/parasitologia , Resistência à Doença/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Soja/parasitologia , Tylenchoidea/patogenicidade , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Bioensaio , Caenorhabditis elegans , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Endotoxinas/metabolismo , Feminino , Engenharia Genética , Proteínas Hemolisinas/metabolismo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Soja/genética , Soja/metabolismo , Tylenchoidea/isolamento & purificação
12.
Toxins (Basel) ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065247

RESUMO

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.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Inseticidas/farmacologia , Mariposas/fisiologia , Animais , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas , Inseticidas/isolamento & purificação , Larva , Ligação Proteica
13.
Toxins (Basel) ; 13(5)2021 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-34065665

RESUMO

Bacillus thuringiensis (Bt), a natural pathogen of different invertebrates, primarily insects, is widely used as a biological control agent. While Bt-based preparations are claimed to be safe for non-target organisms due to the immense host specificity of the bacterium, the growing evidence witnesses the distant consequences of their application for natural communities. For instance, upon introduction to soil habitats, Bt strains can affect indigenous microorganisms, such as bacteria and fungi, and further establish complex relationships with local plants, ranging from a mostly beneficial demeanor, to pathogenesis-like plant colonization. By exerting a direct effect on target insects, Bt can indirectly affect other organisms in the food chain. Furthermore, they can also exert an off-target activity on various soil and terrestrial invertebrates, and the frequent acquisition of virulence factors unrelated to major insecticidal toxins can extend the Bt host range to vertebrates, including humans. Even in the absence of direct detrimental effects, the exposure to Bt treatment may affect non-target organisms by reducing prey base and its nutritional value, resulting in delayed alleviation of their viability. The immense phenotypic plasticity of Bt strains, coupled with the complexity of ecological relationships they can engage in, indicates that further assessment of future Bt-based pesticides' safety should consider multiple levels of ecosystem organization and extend to a wide variety of their inhabitants.


Assuntos
Toxinas de Bacillus thuringiensis/administração & dosagem , Bacillus thuringiensis/patogenicidade , Agentes de Controle Biológico/administração & dosagem , Animais , Toxinas de Bacillus thuringiensis/toxicidade , Agentes de Controle Biológico/toxicidade , Ecossistema , Humanos , Inseticidas/administração & dosagem , Inseticidas/toxicidade , Controle Biológico de Vetores/métodos , Fenótipo
14.
Toxins (Basel) ; 13(5)2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066367

RESUMO

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Bacillus thuringiensis/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Mariposas/fisiologia , Zea mays/genética , Animais , China , Produtos Agrícolas , Resistência a Inseticidas/genética , Larva/genética , Plantas Geneticamente Modificadas/genética
15.
Pestic Biochem Physiol ; 175: 104837, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33993962

RESUMO

Bt protoxins are required to convert to a smaller activated form by insect midgut proteases to exert toxicity against insect pests. Serine protease inhibitors (serpins) play a valuable part in gut protease of insect that hamper digestive proteases activity of insects. Whether the insect serpins induced by Bt protoxin affect the insecticidal activity were rare studied. Here, we identified a serpin-e gene from Helicoverpa armigera, which had potential RCL (Reactive Center Loop) region near the C-terminus like other serpin proteins. It widely expressed in different development stages and in various tissues, but highest expressed in fourth-instar larvae and in larval hemolymph. This Haserpin-e could be induced by Cry1Ac protoxin in vivo and inhibit the midgut proteases to activate Cry1Ac in vitro. Importantly, the functional study indicated it could inhibit the process from Cry1Ac protoxin to activated toxin, and led to the reduction of Cry1Ac insecticide activity to cotton bollworm. Based on our results, we proposed that Haserpin-e involved in the toxicity of Cry1Ac to cotton bollworm by blocking the serine protease to activate the protoxin.


Assuntos
Bacillus thuringiensis , Mariposas , Serpinas , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Resistência a Inseticidas , Larva , Serpinas/genética
16.
Nat Commun ; 12(1): 2791, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990582

RESUMO

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.


Assuntos
Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Variação Genética , Inseticidas/química , Inseticidas/farmacologia , Lipossomos/química , Modelos Moleculares , Controle Biológico de Vetores , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia Estrutural de Proteína
17.
Anal Biochem ; 625: 114222, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33932355

RESUMO

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.


Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais/química , Toxinas de Bacillus thuringiensis/química , Endotoxinas/química , Proteínas Hemolisinas/química , Região Variável de Imunoglobulina/química , Anticorpos de Cadeia Única/química , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Toxinas de Bacillus thuringiensis/imunologia , Toxinas de Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/imunologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Simulação de Acoplamento Molecular , Mariposas , Mutação , Biblioteca de Peptídeos , Conformação Proteica , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo
18.
Arch Insect Biochem Physiol ; 107(3): e21794, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33948968

RESUMO

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Bombyx/efeitos dos fármacos , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Transcriptoma/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bombyx/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo
19.
Int J Biol Macromol ; 183: 1346-1351, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34004200

RESUMO

Anti-idiotypic antibody technique is a new approach for the rapid development of insecticidal protein. In this study, anti-Cry1A polyclonal antibodies were used as antigen to screen the anti-idiotypic antibody that can simulate Cry1A toxins from a phage display human domain antibody (DAB) library. After four rounds of panning, five positive clones that have binding activities with anti-Cry1A polyclonal antibodies were obtained. Indirect competitive ELISA (IC-ELISA) results showed that the positive clone D6 showed significant inhibition for the binding of Cry1A toxins with anti-Cry1A polyclonal antibodies, and the inhibition ratio increased with the increase of D6 content. While, B3, F4, G5, C7 and the controls showed no obvious inhibition to Cry1A toxins. The results suggest that D6 is the "ß" subtype anti-idiotypic antibody, which can simulate Cry1A toxins and competitive binding with anti-Cry1A polyclonal antibodies. Meanwhile, D6 had certain binding activity with the brush border membrane vesicles (BBMV) of p. xylostella, which was the receptor of Cry1A toxins. The results of bioassay showed that D6 had certain insecticidal activity, and the lethal concentration of 50% (LC50) was 976 ng/cm2. This study provides basic materials and experience for the development of Cry toxin simulants.


Assuntos
Toxinas de Bacillus thuringiensis/imunologia , Endotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Biblioteca de Peptídeos , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos
20.
Artigo em Inglês | MEDLINE | ID: mdl-33848588

RESUMO

Development of insect resistance to biopesticides is a current and pertinent global issue. Earlier, it was established that lepidopteran larvae can recover from Bt intoxication via a midgut regenerative response and subsequently generate resistance. Molecular aspects of restoration of the midgut integrity following toxin exposure are emerging recently. In the present study, we bring out the pivotal role of gut arylphorin in mediating the midgut regenerative response following sublethal Bt exposure in Achaea janata. Bt-induced midgut damage was characterized by microscopic analysis using differential interference contrast (DIC) and immunofluorescence (IF). Extensive disruption of brush-border membrane, associated with the underlying cytoskeletal alterations including F-actin, α-actin and ß-tubulin was observed. Single-photon fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) established the metabolic state associated with enhanced stem cell proliferation and migration from the basal side towards the luminal side following the damage. In-silico analysis revealed the phylogenetic relationship of gut arylphorin with closely related insect species and indicated the presence of two different subunits. Transient RNAi knockdown of the arylphorin resulted in diminished expression of mitotic Cyclin B mRNA levels. Human monoclonal Cyclin B antibody cross-reactivity with the Cyclin B located in the stem cells further validate the role of arylphorin as the mitogenic factor responsible for stem cell proliferation and epithelial regeneration. An in-depth understanding of resistance mechanisms will aid in the design of new strategies for the long-term usage and efficacy of Bt technology against pest control.


Assuntos
Toxinas de Bacillus thuringiensis/toxicidade , Proteínas de Insetos/metabolismo , Intestinos , Mariposas/metabolismo , Animais , Bacillus thuringiensis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...