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1.
J Agric Food Chem ; 72(14): 8180-8188, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38556749

RESUMO

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Feminino , Mariposas/genética , Mariposas/metabolismo , Larva/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Longevidade , Sistemas CRISPR-Cas , Endotoxinas/genética , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Resistência a Inseticidas/genética
2.
BMC Genomics ; 25(1): 355, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594617

RESUMO

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.


Assuntos
Bacillus thuringiensis , Mariposas , Praguicidas , Animais , Larva/genética , Larva/metabolismo , Soja/genética , Endotoxinas/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Controle Biológico de Vetores/métodos , Mariposas/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Cromossomos/metabolismo , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Resistência a Inseticidas/genética
3.
Lett Appl Microbiol ; 77(3)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429983

RESUMO

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.


Assuntos
Bacillus thuringiensis , Inseticidas , Inseticidas/análise , Endotoxinas/análise , Endotoxinas/química , Resíduos/análise , Toxinas de Bacillus thuringiensis/análise , Proteínas de Bactérias/química , Proteínas Hemolisinas , Eletroforese em Gel de Poliacrilamida
4.
Biomolecules ; 14(3)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38540692

RESUMO

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Toxinas de Bacillus thuringiensis , Bombyx , Proteínas Hemolisinas , Animais , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bombyx/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Endotoxinas , Insetos/metabolismo , Proteínas de Bactérias/metabolismo
5.
Pestic Biochem Physiol ; 199: 105777, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38458684

RESUMO

The fall armyworm (Spodoptera frugiperda) is a major global pest causing severe damage to various crops, especially corn. Transgenic corn producing the Cry1F pesticidal protein from the bacterium Bacillus thuringiensis (Cry1F corn) showed effectiveness in controlling this pest until S. frugiperda populations at locations in North and South America evolved practical resistance. The mechanism for practical resistance involved disruptive mutations in an ATP binding cassette transporter subfamily C2 gene (SfABCC2), which serves as a functional Cry1F receptor in the midgut cells of susceptible S. frugiperda. The SfABCC2 protein contains two transmembrane domains (TMD1 and TMD2), each with a cytosolic nucleotide (ATP) binding domain (NBD1 and NBD2, respectively). Previous reports have demonstrated that disruptive mutations in TMD2 were linked with resistance to Cry1F, yet whether the complete SfABCC2 structure is needed for receptor functionality or if a single TMD-NBD protein can serve as functional Cry1F receptor remains unknown. In the present study, we separately expressed TMD1 and TMD2 with their corresponding NBDs in cultured insect cells and tested their Cry1F receptor functionality. Our results show that the complete SfABCC2 structure is required for Cry1F receptor functionality. Moreover, binding competition assays revealed that Cry1F specifically bound to SfABCC2, whereas neither SfTMD1-NBD1 nor SfTMD2-NBD2 exhibited any significant binding. These results provide insights into the molecular mechanism of Cry1F recognition by SfABCC2 in S. frugiperda, which could facilitate the development of more effective insecticidal proteins.


Assuntos
Bacillus thuringiensis , Endotoxinas , Animais , Spodoptera , Endotoxinas/genética , Resistência a Inseticidas/genética , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacillus thuringiensis/metabolismo , Zea mays , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas/genética
6.
Int J Biol Macromol ; 263(Pt 1): 130271, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38373570

RESUMO

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides.


Assuntos
Bacillus thuringiensis , Inseticidas , MicroRNAs , Mariposas , Animais , Mariposas/metabolismo , Larva/genética , Endotoxinas/genética , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/química , Inseticidas/farmacologia , Inseticidas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
7.
Toxins (Basel) ; 16(2)2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38393170

RESUMO

Paralipsa gularis (Zeller) is a storage pest; however, in recent years it has evolved into a considerable maize pest during the late growth stage in the border region between China and other Southeast Asian countries. Bt transgenic insect-resistant maize is an effective measure in controlling a wide range of lepidopteran pests, but there is a lack of research on the toxic effects of storage pests. We tested the toxicity of Bt-Cry1Ab, Vip3Aa, and their complex proteins against P. gularis via bioassay and investigated the efficiency of Bt-(Cry1Ab+Vip3Aa) maize in controlling P. gularis during the late growth stage of maize in the period 2022-2023. The bioassay results show that the susceptibilities of P. gularis to the two Bt proteins and their complex proteins were significantly different. The LC50 values of DBNCry1Ab ("DBN9936" event), DBNVip3Aa ("DBN9501" event), DBN Cry1Ab+Vip3Aa ("DBN3601T" event), and Syngenta Cry1Ab+Vip3Aa ("Bt11" event × "MIR162" event) were 0.038 µg/g, 0.114 µg/g, 0.110 µg/g, and 0.147 µg/g, and the GIC50 values were 0.014 µg/g, 0.073 µg/g, 0.027 µg/g, and 0.026 µg/g, respectively. Determination of the expression content of the insecticidal protein in different tissues of Bt-(Cry1Ab+Vip3Aa) maize shows that the total Bt protein content in different tissues was in the following order: stalk > bract > cob > kernel. However, the bioassay results show that the mortalities of P. gularis feeding on Bt-(Cry1Ab+Vip3Aa) maize in different tissues at different growth stages were all above 93.00%. The field trial indicates that the occurrence density of larvae and plant damage rate for conventional maize were 422.10 individuals/100 plants and 94.40%, respectively, whereas no larvae were found on Bt-(Cry1Ab+Vip3Aa) maize. In summary, this study implies that Bt-(Cry1Ab+Vip3Aa) maize has a high potential for control of P. gularis, providing a new technical measure for the management of the pest.


Assuntos
Bacillus thuringiensis , Lepidópteros , Humanos , Animais , Zea mays/genética , Zea mays/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/genética , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/genética , Controle Biológico de Vetores/métodos , Lepidópteros/metabolismo , Larva
8.
Toxins (Basel) ; 16(2)2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38393166

RESUMO

Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura. To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II-V.


Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/metabolismo , Lipossomos/metabolismo , Conformação Proteica em alfa-Hélice , Inseticidas/química , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Larva/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo
9.
Curr Microbiol ; 81(3): 80, 2024 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-38281302

RESUMO

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.


Assuntos
Aedes , Bacillus thuringiensis , Culex , Culicidae , Animais , Bacillus thuringiensis/metabolismo , Culicidae/metabolismo , Endotoxinas/genética , Endotoxinas/toxicidade , Endotoxinas/química , Toxinas de Bacillus thuringiensis , Sequência de Aminoácidos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Larva , Cátions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/química
10.
Pestic Biochem Physiol ; 198: 105744, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38225087

RESUMO

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , 60627 , Endotoxinas/toxicidade , Endotoxinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Escherichia coli , Toxinas de Bacillus thuringiensis/metabolismo , Mariposas/genética , Mariposas/metabolismo , Larva/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/metabolismo , Bacillus thuringiensis/metabolismo , Resistência a Inseticidas
11.
J Agric Food Chem ; 72(2): 1321-1329, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38175929

RESUMO

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.


Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/química , Inseticidas/química , Endotoxinas/genética , Endotoxinas/toxicidade , Endotoxinas/química , Domínios Proteicos , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/toxicidade , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/química , Larva/metabolismo
12.
Int J Biol Macromol ; 254(Pt 3): 127985, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37949263

RESUMO

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.


Assuntos
Bacillus thuringiensis , Bacillus thuringiensis/química , Endotoxinas/química , Toxinas de Bacillus thuringiensis/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas Hemolisinas/química , Chaperonas Moleculares/metabolismo
13.
Adv Sci (Weinh) ; 11(6): e2307650, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38087901

RESUMO

Bioinsecticides and transgenic crops based on the bacterial pathogen Bacillus thuringiensis (Bt) can effectively control diverse agricultural insect pests, nevertheless, the evolution of resistance without obvious fitness costs has seriously eroded the sustainable use of these Bt products. Recently, it has been discovered that an increased titer of juvenile hormone (JH) favors an insect host (Plutella xylostella) to enhance fitness whilst resisting the Bt pathogen, however, the underlying regulatory mechanisms of the increased JH titer are obscure. Here, the involvement of N6 -methyladenosine (m6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m6 A methyltransferase subunit genes, PxMettl3 and PxMettl14, repress the expression of a key JH-degrading enzyme JH esterase (JHE) to induce an increased JH titer, mitigating the fitness costs associated with a robust defense against the Bt pathogen. This study identifies an as-yet uncharacterized m6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m6 A methylation atlas in insects, which further expands the functional landscape of m6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Mariposas/genética , Mariposas/metabolismo , RNA/metabolismo , Epigênese Genética/genética , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/farmacologia , Toxinas de Bacillus thuringiensis/metabolismo , Insetos , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Hormônios Juvenis/metabolismo , Metilação
14.
Int J Biol Macromol ; 257(Pt 1): 128654, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38065453

RESUMO

The bacterium Bacillus thuringiensis (Bt) is the most economically successful biopesticide to date, and Bt insecticidal proteins are produced in transgenic crops for pest control. However, relevant details in the Bt-mediated killing process remain undefined. In our previous research, we observed reduced larval susceptibility to Bt Cry1Ca in Chilo suppressalis, a major rice pest in China, after gut microbiota elimination. Here, we tested the hypothesis that gut microbiota, particularly abundant Enterococcus spp., influences C. suppressalis susceptibility to Cry1Ca. We isolated and identified four Enterococcus spp. from C. suppressalis gut microbiota and evaluated their impact on Cry1Ca toxicity. Among the four Enterococcus spp. identified, three of them (E. casseliflavus, E. faecalis, and E. mundtii) dramatically increased larval mortality when introduced in axenic C. suppressalis challenged with Cry1Ca. Gut epithelial damage by Cry1Ca promoted the translocation of Enterococcus spp. from the gut lumen into the hemocoel, where they proliferated and induced larval melanization and hemocyte apoptosis. Our combined findings demonstrate that the presence of specific gut microbiota can greatly affect susceptibility to Cry1Ca through melanization and apoptosis of hemocytes. Better understanding of the Bt intoxication process guides the development of bio-enhancers for Bt-based microbial biopesticides and potential improvement of transgenic crops.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Oryza , Animais , Enterococcus , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Proteínas Hemolisinas/metabolismo , Mariposas/genética , Larva , Inseticidas/farmacologia , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/metabolismo , Animais Geneticamente Modificados , Oryza/genética
15.
Toxicon ; 238: 107588, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38147939

RESUMO

Pest insects pose a heavy burden on global agricultural industries with small molecule insecticides being predominantly used for their control. Unwanted side effects and resistance development plagues most small molecule insecticides such as the neonicotinoids, which have been reported to be harmful to honeybees. Bioinsecticides like Bacillus thuringiensis (Bt) toxins can be used as environmentally-friendly alternatives. Arachnid venoms comprise another promising source of bioinsecticides, containing a multitude of selective and potent insecticidal toxins. Unfortunately, no standardised insect models are currently available to assess the suitability of insecticidal agents under laboratory conditions. Thus, we aimed to develop a laboratory model that closely mimics field conditions by employing a leaf disk assay (LDA) for oral application of insecticidal agents in a bioassay tray format. Neonate larvae of the cotton bollworm (Helicoverpa armigera) were fed with soybean (Glycine max) leaves that were treated with different insecticidal agents. We observed dose-dependent insecticidal effects for Bt toxin and the neonicotinoid insecticide imidacloprid, with imidacloprid exhibiting a faster response. Furthermore, we identified several insecticidal arachnid venoms that were active when co-applied with sub-lethal doses of Bt toxin. We propose the H. armigera LDA as a suitable tool for assessing the insecticidal effects of insecticidal agents against lepidopterans.


Assuntos
Venenos de Artrópodes , Bacillus thuringiensis , Inseticidas , Mariposas , Neonicotinoides , Nitrocompostos , Toxinas Biológicas , Humanos , Recém-Nascido , Animais , Inseticidas/toxicidade , Soja , 60627 , Toxinas de Bacillus thuringiensis/farmacologia , Larva , Insetos , Toxinas Biológicas/farmacologia , Venenos de Artrópodes/farmacologia , Bioensaio , Folhas de Planta , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/toxicidade , Endotoxinas , Controle Biológico de Vetores , Resistência a Inseticidas
16.
Pestic Biochem Physiol ; 197: 105658, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38072533

RESUMO

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Endotoxinas/genética , Endotoxinas/farmacologia , Endotoxinas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/metabolismo , Resistência a Inseticidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/metabolismo , Mariposas/genética , Mariposas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/metabolismo , Gossypium/metabolismo , Larva/genética
17.
Int J Mol Sci ; 24(23)2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-38069132

RESUMO

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.


Assuntos
Toxinas de Bacillus thuringiensis , Bacillus thuringiensis , Animais , Furilfuramida/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Bacillus thuringiensis/química , Proteínas de Bactérias/metabolismo , Larva
18.
Braz J Biol ; 83: e277899, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38126646

RESUMO

Transgenic Bt soybean plants have been developed to control insect pests, such as Anticarsia gemmatalis and Chrysodeixis includens. This objective has been achieved successfully; however, recently, some authors claimed that Bt soybean plants have been more susceptible than non-Bt soybean to Bemisia tabaci MEAM1. In addition, it is unknown whether Bt soybean plants infested by B. tabaci become less resistant to target pests. Therefore, this study aimed to evaluate: (i) whether the previous infestation with B. tabaci can compromise Bt and non-Bt soybean resistance to C. includens; (ii) the effects of B. tabaci infestations on Bt and non-Bt soybean plant growth; and (iii) whether B. tabaci feeding reduces contents of chlorophyll and carotenoids of soybean plants. Bt and non-Bt soybean plants pre-infested with B. tabaci showed no changes in resistance to C. includens. Bt soybean plants infested with B. tabaci showed a lower plant height than uninfested plants. Differently, non-Bt soybean plants exhibited no reduction in plant growth due to B. tabaci feeding. Bt soybean plants suffered a reduction in dry matter only under double infestation (B. tabaci and C. includens), while non-Bt soybean plants experienced reduction in dry matter when infested with B. tabaci and C. includens or by C. includens only. B. tabaci feeding did not alter contents of chlorophyll and carotenoids, and perhaps the reduction in plant growth was related to salivary toxins. Concluding, both Bt and non-Bt soybean plants were susceptible to B. tabaci feeding, evidencing necessity of developing soybean cultivars resistant to B. tabaci.


Assuntos
Soja , Mariposas , Animais , Endotoxinas/farmacologia , Toxinas de Bacillus thuringiensis/farmacologia , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Carotenoides/farmacologia , Clorofila
19.
Pestic Biochem Physiol ; 196: 105596, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37945246

RESUMO

Cry and Vip3 proteins are both pore-forming toxins produced by Bacillus thuringiensis that show synergistic insecticidal activity against different insect pests. However, the synergistic effect of Cry and Vip3 proteins on the midgut in target insects is still unclear. In this study, faster and more serious damage was observed after treatment with both Cry9A and Vip3A proteins in the Chilo suppressalis midgut compared to single-protein treatment. Through RNA sequencing, midgut transcriptomic comparison was performed between dual- and single-protein treatments according to midgut injury. After 6 h, 609 differentially expressed genes were found with the combined Cry9A and Vip3A treatments, which was much more than that in the single treatment, corresponding to faster and more serious damage. These genes were mainly enriched in similar pathways, such as lipid metabolic, oxidation-reduction and carbohydrate metabolic process, peptide secretion and cell-cell adhesion; however, the number and expression level of differentially expressed genes are increased. For specific genes significantly regulated by induction of Cry9A and Vip3A, lipases, phospholipid scramblase, probable tape measure protein and arylsulfatase J were significantly downregulated after 6 h treatment. In addition, regular genes related to the activation and receptor binding of B. thuringiensis toxins were differentially regulated, such as ATP-binding cassette subfamily G member 1 and serine protease. Validation with RT-qPCR showed agreement with the sequencing results. Overall, our results support that stronger and faster midgut responses at the cellular and transcriptional levels are induced by the synergistic toxicity of Cry9A and Vip3A in C. suppressalis.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Larva , Endotoxinas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/farmacologia , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/metabolismo
20.
Biochem Biophys Res Commun ; 685: 149144, 2023 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-37922785

RESUMO

In-cell protein crystals which spontaneously crystallize in living cells, have recently been analyzed in investigations of their structures and biological functions. The crystals have been challenging to analyze structurally because of their small size. Therefore, the number of in-cell protein crystals in which the native structure has been determined is limited because most of the structures of in-cell crystals have been determined by recrystallization after dissolution. Some proteins have been reported to form intermolecular disulfide bonds in natural protein crystals that stabilize the crystals. Here, we focus on Cry1Aa, a cysteine-rich protein that crystallizes in Bacillus thuringiensis (Bt) and forms disulfide bonds. Previously, the full-length structure of 135 kDa Cry1Ac, which is the same size as Cry1Aa, was determined by recrystallization of dissolved protein from crystals purified from Bt cells. However, the formation of disulfide bonds has not been investigated because it was necessary to replace cysteine residues to prevent aggregation of the soluble protein. In this work, we succeeded in direct X-ray crystallographic analysis using crystals purified from Bt cells and characterized the cross-linked network of disulfide bonds within Cry1Aa crystals.


Assuntos
Bacillus thuringiensis , Bacillus thuringiensis/metabolismo , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Cisteína/metabolismo , Proteínas de Bactérias/metabolismo , Dissulfetos/metabolismo , Proteínas Hemolisinas/metabolismo
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