ABSTRACT
The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Insect Proteins , Insecticides , Larva , Moths , Animals , Bacillus thuringiensis Toxins/metabolism , Bacillus thuringiensis Toxins/toxicity , Bacillus thuringiensis Toxins/chemistry , Endotoxins/metabolism , Endotoxins/genetics , Endotoxins/toxicity , Hemolysin Proteins/metabolism , Hemolysin Proteins/pharmacology , Hemolysin Proteins/toxicity , Hemolysin Proteins/genetics , Moths/metabolism , Moths/drug effects , Moths/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/toxicity , Insect Proteins/metabolism , Insect Proteins/genetics , Larva/metabolism , Larva/drug effects , Larva/growth & development , Larva/genetics , Insecticides/toxicity , Insecticides/pharmacology , Insecticides/chemistry , Bacillus thuringiensis/chemistry , Bacillus thuringiensis/metabolism , Bacillus thuringiensis/genetics , Insecticide Resistance/genetics , Pest Control, Biological , Helicoverpa armigeraABSTRACT
Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.
Subject(s)
Bacillus thuringiensis Toxins , Bacillus thuringiensis , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Humans , Endotoxins/chemistry , Endotoxins/genetics , Endotoxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Bacillus thuringiensis Toxins/metabolism , Hemolysin Proteins/chemistry , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Hemolysin Proteins/pharmacology , Animals , Insecticides/chemistry , Insecticides/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Aedes/drug effects , Aedes/genetics , Cell Line, TumorABSTRACT
Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Moths , Animals , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Bacillus thuringiensis Toxins/metabolism , Bacterial Proteins/metabolism , Moths/metabolism , Moths/microbiology , Binding Sites , Bacillus thuringiensis/metabolism , Pest Control, Biological , Protein Domains , Helicoverpa armigeraABSTRACT
It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.
Subject(s)
Aedes , Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Proteolysis , Animals , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Hemolysin Proteins/metabolism , Bacterial Proteins/metabolism , Pest Control, Biological , Bacillus thuringiensisABSTRACT
The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Animals , Bacillus thuringiensis Toxins/metabolism , Endotoxins/metabolism , Endotoxins/genetics , Endotoxins/chemistry , Hemolysin Proteins/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Proteins/chemistry , Moths/metabolism , Moths/genetics , Bacillus thuringiensis/metabolism , Bacillus thuringiensis/genetics , Molecular Docking Simulation , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/chemistryABSTRACT
By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.
Subject(s)
Multidrug Resistance-Associated Protein 2 , Animals , Bacillus thuringiensis Toxins/metabolism , Binding Sites , Bombyx/metabolism , Bombyx/genetics , Endotoxins/metabolism , Endotoxins/chemistry , Hemolysin Proteins/metabolism , Hemolysin Proteins/chemistry , Insect Proteins/metabolism , Insect Proteins/genetics , Insect Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/chemistry , Protein BindingABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Moths , Animals , Female , Moths/genetics , Moths/metabolism , Larva/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Longevity , CRISPR-Cas Systems , Endotoxins/genetics , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Insecticide Resistance/geneticsABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Endotoxins , Animals , Spodoptera , Endotoxins/genetics , Insecticide Resistance/genetics , Bacillus thuringiensis Toxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacillus thuringiensis/metabolism , Zea mays , Hemolysin Proteins/genetics , Plants, Genetically Modified/geneticsABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Insecticides , MicroRNAs , Moths , Animals , Moths/metabolism , Larva/genetics , Endotoxins/genetics , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Bacillus thuringiensis/chemistry , Insecticides/pharmacology , Insecticides/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Hemolysin Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Lepidoptera , Humans , Animals , Zea mays/genetics , Zea mays/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Bacterial Proteins/toxicity , Bacterial Proteins/genetics , Hemolysin Proteins/toxicity , Hemolysin Proteins/genetics , Pest Control, Biological/methods , Lepidoptera/metabolism , LarvaABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Insecticides , Animals , Bacillus thuringiensis/metabolism , Liposomes/metabolism , Protein Conformation, alpha-Helical , Insecticides/chemistry , Bacillus thuringiensis Toxins/metabolism , Bacterial Proteins/metabolism , Larva/metabolism , Endotoxins/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolismABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Insecticides , Moths , Animals , Helicoverpa armigera , Endotoxins/toxicity , Endotoxins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Escherichia coli , Bacillus thuringiensis Toxins/metabolism , Moths/genetics , Moths/metabolism , Larva/metabolism , Insecticides/toxicity , Insecticides/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/toxicity , Hemolysin Proteins/metabolism , Bacillus thuringiensis/metabolism , Insecticide ResistanceABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Bacillus thuringiensis/chemistry , Endotoxins/chemistry , Bacillus thuringiensis Toxins/metabolism , Escherichia coli/metabolism , Bacterial Proteins/chemistry , Hemolysin Proteins/chemistry , Molecular Chaperones/metabolismABSTRACT
BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.
Subject(s)
Bacillus thuringiensis , Lepidoptera , Moths , Oryza , Animals , Lepidoptera/genetics , Oryza/genetics , Oryza/metabolism , Endotoxins/pharmacology , Bacillus thuringiensis Toxins/metabolism , Bacillus thuringiensis Toxins/pharmacology , Bacterial Proteins/pharmacology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Hemolysin Proteins/metabolism , Bacillus thuringiensis/genetics , Pest Control, Biological/methodsABSTRACT
Intensification in agriculture affects many insect species, including butterflies. Insect-resistant crops, such as Bt (Bacillus thuringiensis) maize, which produces a toxin active against Lepidoptera, are an alternative to insecticide sprays. Genetically modified crops are regulated in most countries and require an environmental risk assessment. In the European Union, such assessments include the use of simulation models to predict the effects on nontarget Lepidoptera (NTL). To support the assessment of protected NTL, we extended an individual-based, stochastic, spatially explicit mathematical model (LepiX) to include a wider range of exposure scenarios, a species-sensitivity distribution, and an option for repeated exposure of individuals. We applied the model to transgenic maize DAS-1507, which expresses a high concentration of Bt toxin in pollen that may be consumed by NTL larvae on their host plants nearby. Even in the most conservative scenario without repeated exposure, mortality estimates for highly sensitive species ranged from 41% to 6% at distances of 10-1000 m from the nearest maize field. Repeated exposure can cause additional mortality and thus is relevant for the overall risk assessment. Uncertainties in both exposure and ecotoxicity estimates strongly influenced the predicted mortalities. Care should be taken to include these uncertainties in the model scenarios used for decision-making. In accordance with other modeling results, our simulations demonstrated that mean mortality may not be safe for protected species. With its high pollen expression, DAS-1507 maize may pose risks to sensitive and protected butterfly and moth species that may be difficult to manage. High expression of Bt toxin in pollen is unnecessary for controlling target pests. Consequently, we suggest that Bt maize with high pollen expression not be cultivated in regions where protected butterflies are to be conserved.
La intensificación en la agricultura afecta a muchas especies de insectos, incluyendo a las mariposas. Los cultivos resistentes a los insectos, como el maíz Bt (Bacillus thuringiensis), el cual produce una toxina activa contra los lepidópteros, son una alternativa a los insecticidas. Los cultivos genéticamente modificados (GM) están regulados en la mayoría de los países y requieren de una evaluación de riesgo ambiental. En la Unión Europea (EU), dichas evaluaciones incluyen el uso de modelos de simulación para pronosticar los efectos sobre los lepidópteros no objetivo (LNO). Para apoyar a la evaluación de LNO protegidos, extendimos un modelo matemático espacialmente explícito, estocástico y basado en el individuo (LepiX) para incluir una mayor gama de escenarios de exposición, una distribución de la sensibilidad de las especies y una opción para la exposición repetida de los individuos. Aplicamos el modelo al maíz transgénico DAS1507, el cual expresa una alta concentración de toxina Bt en el polen que puede ser consumido por las larvas de LNO en una planta hospedera cercana. Incluso en el escenario más conservador sin una exposición repetida, las estimaciones de mortalidad para las especies altamente sensibles variaron entre el 41% y el 6% en distancias de 101000 m a partir del campo de maíz más cercano. La exposición repetida puede causar mortalidad adicional y por lo tanto es relevante para la evaluación general del riesgo. La incertidumbre en las estimaciones de la exposición y la ecotoxicidad influyeron fuertemente sobre la mortalidad pronosticada. Se debe tener cuidado de incluir estas incertidumbres en los escenarios modelados usados para la toma de decisiones. De acuerdo con los resultados de otros modelos, nuestras simulaciones demostraron que la mortalidad media podría no ser segura para las especies protegidas. Con su alta producción de polen, el maíz DAS1507 podría representar un riesgo difícil de manejar para las especies de mariposas y polillas sensibles y protegidas. No se necesita una expresión elevada de la toxina Bt en el polen para controlar a las plagas. En consecuencia, sugerimos que no se cultive el maíz Bt con una alta producción de polen en las regiones en donde se busca conservar a las mariposas protegidas. Presión del maíz resistente a insectos sobre mariposas y polillas protegidas.
Subject(s)
Butterflies , Moths , Animals , Butterflies/genetics , Moths/genetics , Zea mays/genetics , Zea mays/metabolism , Bacillus thuringiensis Toxins/metabolism , Crops, Agricultural , Plants, Genetically Modified/genetics , Conservation of Natural Resources , Insecta , Larva/geneticsABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Insecticides , Moths , Oryza , Animals , Enterococcus , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Pest Control, Biological , Plants, Genetically Modified , Hemolysin Proteins/metabolism , Moths/genetics , Larva , Insecticides/pharmacology , Bacterial Proteins/toxicity , Bacterial Proteins/metabolism , Animals, Genetically Modified , Oryza/geneticsABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Moths , Animals , Moths/genetics , Moths/metabolism , RNA/metabolism , Epigenesis, Genetic/genetics , Endotoxins/genetics , Endotoxins/metabolism , Endotoxins/pharmacology , Bacillus thuringiensis Toxins/metabolism , Insecta , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Juvenile Hormones/metabolism , MethylationABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Moths , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Helicoverpa armigera , Endotoxins/genetics , Endotoxins/pharmacology , Endotoxins/metabolism , ATP-Binding Cassette Transporters/genetics , Bacillus thuringiensis Toxins/metabolism , Insecticide Resistance/genetics , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Bacterial Proteins/metabolism , Moths/genetics , Moths/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Hemolysin Proteins/metabolism , Gossypium/metabolism , Larva/geneticsABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Bacillus thuringiensis/metabolism , Endotoxins/metabolism , Bacillus thuringiensis Toxins/metabolism , Cysteine/metabolism , Bacterial Proteins/metabolism , Disulfides/metabolism , Hemolysin Proteins/metabolismABSTRACT
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.