ABSTRACT
Bacillus thuringiensis is the most widely used biopesticide, targets a diversity of insect pests belonging to several orders. However, information regarding the B. thuringiensis strains and toxins targeting Zeugodacus cucurbitae is very limited. Therefore, in the present study, we isolated and identified five indigenous B. thuringiensisstrains toxic to larvae of Z. cucurbitae. However, of five strains NBAIR BtPl displayed the highest mortality (LC50 = 37.3 µg/mL) than reference strain B. thuringiensis var. israelensis (4Q1) (LC50 = 45.41 µg/mL). Therefore, the NBAIR BtPl was considered for whole genome sequencing to identify the cry genes present in it. Whole genome sequencing of our strain revealed genome size of 6.87 Mb with 34.95% GC content. Homology search through the BLAST algorithm revealed that NBAIR BtPl is 99.8% similar to B. thuringiensis serovar tolworthi, and gene prediction through Prokka revealed 7406 genes, 7168 proteins, 5 rRNAs, and 66 tRNAs. BtToxin_Digger analysis of NBAIR BtPl genome revealed four cry gene families: cry1, cry2, cry8Aa1, and cry70Aa1. When tested for the presence of these four cry genes in other indigenous strains, results showed that cry70Aa1 was absent. Thus, the study provided a basis for predicting cry70Aa1 be the possible reason for toxicity. In this study apart from novel genes, we also identified other virulent genes encoding zwittermicin, chitinase, fengycin, and bacillibactin. Thus, the current study aids in predicting potential toxin-encoding genes responsible for toxicity to Z. cucurbitae and thus paves the way for the development of B. thuringiensis-based formulations and transgenic crops for management of dipteran pests.
Subject(s)
Bacillus thuringiensis , Bacterial Proteins , Genome, Bacterial , Whole Genome Sequencing , Bacillus thuringiensis/genetics , Animals , Bacterial Proteins/genetics , Bacillus thuringiensis Toxins/genetics , Endotoxins/genetics , Pest Control, Biological , Tephritidae/genetics , Tephritidae/microbiology , Hemolysin Proteins/genetics , Larva/genetics , PhylogenyABSTRACT
Exopolysaccharides (EPSs) are carbohydrate polymers that are synthesized and secreted into the extracellular during the growth of microorganisms. Bacillus thuringiensis (Bt) is a type of entomopathogenic bacterium, that produces various insecticidal proteins and EPSs. In our previous study, the EPSs produced by Bt strains were first found to enhance the toxicity of insecticidal crystal proteins against Plutella xylostella. However, the response of the intestinal bacterial communities of P. xylostella under the action of EPSs is still unelucidated. In this study, 16S rRNA amplicon sequencing was used to characterize the intestinal bacterial communities in P. xylostella treated with EPSs alone, Cry1Ac protoxin alone, and both the Cry1Ac protoxin and EPSs. Compared with the control group, alpha diversity indices, the Chao1 and ACE indices were significantly altered after treatment with EPSs alone, and no significant difference was observed between the groups treated with Cry1Ac protoxin alone and Cry1Ac protoxin + EPSs. However, compared with the gut bacterial community feeding on Cry1Ac protoxin alone, the relative abundance of 31 genera was significantly changed in the group treated with Cry1Ac protoxin and EPSs. The intestinal bacteria, through the oral of Cry1Ac protoxin and EPSs, significantly enhanced the toxicity of the Cry1Ac protoxin towards the axenic P. xylostella. In addition, the relative abundance of the 16S rRNA gene in the chloroplasts of Brassica campestris decreased after adding EPSs. Taken together, these results show the vital contribution of the gut microbiota to the Bt strain-killing activity, providing new insights into the mechanism of the synergistic insecticidal activity of Bt proteins and EPSs.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Gastrointestinal Microbiome , Hemolysin Proteins , Moths , Animals , Gastrointestinal Microbiome/drug effects , Endotoxins/genetics , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Moths/drug effects , Moths/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , RNA, Ribosomal, 16S/genetics , Bacillus thuringiensis/genetics , Insecticides/pharmacologyABSTRACT
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
Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.
Subject(s)
Bacillus thuringiensis , Bacterial Proteins , Insecticides , Proteomics , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Proteomics/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Insecticides/pharmacology , Whole Genome Sequencing/methods , Genome, Bacterial , Endotoxins/genetics , Bacillus thuringiensis Toxins , Genomic Islands , Proteome , Plasmids/genetics , Tandem Mass Spectrometry , Animals , Hemolysin Proteins/geneticsABSTRACT
BACKGROUND: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines. METHODS: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages. RESULTS: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues. CONCLUSIONS: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops.
Subject(s)
Bacterial Proteins , Endotoxins , Oryza , Plants, Genetically Modified , Oryza/genetics , Oryza/parasitology , Plants, Genetically Modified/genetics , Animals , Endotoxins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hemolysin Proteins/genetics , Bacillus thuringiensis Toxins/genetics , Bacillus thuringiensis/genetics , Moths/genetics , Gene Expression Regulation, Plant , Pest Control, Biological/methodsABSTRACT
Bacillus thuringiensis (Bt) toxins are potential alternatives to synthetic insecticides for the control of lepidopteran pests. However, the evolution of resistance in some insect pest populations is a threat and can reduce the effectiveness of Bt toxins. In this review, we summarize the results of 161 studies from 20 countries reporting field and laboratory-evolved resistance, cross-resistance, and inheritance, mechanisms, and fitness costs of resistance to different Bt toxins. The studies refer mainly to insects from the United States of America (70), followed by China (31), Brazil (19), India (12), Malaysia (9), Spain (3), and Australia (3). The majority of the studies revealed that most of the pest populations showed susceptibility and a lack of cross-resistance to Bt toxins. Factors that delay resistance include recessive inheritance of resistance, the low initial frequency of resistant alleles, increased fitness costs, abundant refuges of non-Bt, and pyramided Bt crops. The results of field and laboratory resistance, cross-resistance, and inheritance, mechanisms, and fitness cost of resistance are advantageous for predicting the threat of future resistance and making effective strategies to sustain the effectiveness of Bt crops.
Subject(s)
Bacillus thuringiensis Toxins , Bacillus thuringiensis , Insecticide Resistance , Pest Control, Biological , Animals , Insecticide Resistance/genetics , Bacillus thuringiensis/genetics , Lepidoptera/drug effects , Genetic Fitness , Insecticides/pharmacology , Endotoxins/geneticsABSTRACT
Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Insecticide Resistance , Spodoptera , Transcriptome , Spodoptera/drug effects , Spodoptera/genetics , Animals , Endotoxins/genetics , Endotoxins/pharmacology , Hemolysin Proteins/genetics , Hemolysin Proteins/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Insecticide Resistance/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/parasitology , Zea mays/genetics , Zea mays/parasitology , Gene Expression ProfilingABSTRACT
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
The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.
Subject(s)
Aedes , Alkaline Phosphatase , Bacillus thuringiensis Toxins , Bacterial Proteins , CRISPR-Cas Systems , Cadherins , Endotoxins , Hemolysin Proteins , Animals , Aedes/genetics , Aedes/drug effects , Alkaline Phosphatase/metabolism , Alkaline Phosphatase/genetics , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Endotoxins/genetics , Endotoxins/metabolism , Female , Cadherins/genetics , Cadherins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Insecticide Resistance/genetics , Gene Knockout Techniques , Larva/genetics , Larva/growth & development , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Male , Insecticides/pharmacologyABSTRACT
BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.
Subject(s)
Arabidopsis , Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Hemolysin Proteins , Plants, Genetically Modified , Plants, Genetically Modified/genetics , Arabidopsis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hemolysin Proteins/genetics , Animals , Endotoxins/genetics , Promoter Regions, Genetic/genetics , Bacillus thuringiensis/genetics , Moths/genetics , Brassica/genetics , Pest Control, Biological/methods , Insecticides/pharmacologyABSTRACT
Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops.
Subject(s)
Glycine max , Plants, Genetically Modified , Glycine max/genetics , Glycine max/parasitology , Glycine max/growth & development , Animals , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Endotoxins/genetics , Brazil , Pest Control, Biological , Hemolysin Proteins/genetics , Crops, Agricultural/genetics , Insecta/genetics , Insecta/pathogenicity , Lepidoptera/pathogenicity , Lepidoptera/genetics , Bacterial Proteins/genetics , Bacillus thuringiensis Toxins/geneticsABSTRACT
The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb-Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana. The virulence of Bb-Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT50) for A. aegypti larvae infected with Bb-Cyt1Aa decreased by 33.3% at a concentration of 1 × 108 conidia/mL and by 22.2% at 1 × 107 conidia/mL. The LT50 for A. aegypti adults infected with Bb-Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 108 conidia/mL and by 33.3% at 1 × 107 conidia/mL. Likewise, the LT50 for A. aegypti adults infected with Bb-Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb-Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus, when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control. IMPORTANCE: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus, thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control.
Subject(s)
Aedes , Bacillus thuringiensis Toxins , Bacillus thuringiensis , Beauveria , Endotoxins , Hemolysin Proteins , Larva , Mosquito Control , Pest Control, Biological , Animals , Beauveria/genetics , Beauveria/pathogenicity , Beauveria/metabolism , Aedes/microbiology , Mosquito Control/methods , Bacillus thuringiensis Toxins/genetics , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Endotoxins/genetics , Endotoxins/metabolism , Pest Control, Biological/methods , Larva/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence/genetics , Spores, Fungal/genetics , Insecticides/pharmacology , Insecticides/metabolismABSTRACT
Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.
Subject(s)
Agrobacterium tumefaciens , Coffea , Coffea/genetics , Agrobacterium tumefaciens/genetics , CRISPR-Cas Systems , Plants, Genetically Modified/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacillus thuringiensis/genetics , Endotoxins/genetics , Bacillus thuringiensis Toxins , Gene Editing/methods , Hemolysin Proteins/genetics , Gene Expression Regulation, Plant , Transformation, Genetic , Coffee/geneticsABSTRACT
The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.
Subject(s)
Bacillus thuringiensis Toxins , Bacterial Proteins , Endotoxins , Gossypium , Hemolysin Proteins , Larva , Plants, Genetically Modified , Weevils , Gossypium/genetics , Gossypium/parasitology , Animals , Weevils/genetics , Plants, Genetically Modified/genetics , Endotoxins/genetics , Endotoxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Hemolysin Proteins/pharmacology , Larva/drug effects , Bacillus thuringiensis/genetics , Pest Control, BiologicalABSTRACT
Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.
Subject(s)
Bacillus thuringiensis Toxins , Hemolysin Proteins , Insecticide Resistance , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins , Promoter Regions, Genetic , Spodoptera , Transcription Factors , Animals , Spodoptera/genetics , Spodoptera/drug effects , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Insecticide Resistance/genetics , Hemolysin Proteins/genetics , Promoter Regions, Genetic/genetics , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Endotoxins/genetics , Gene Expression Regulation/drug effects , Larva/drug effects , Larva/geneticsABSTRACT
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
BACKGROUND: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density-dependent mortality. We conducted a 2-year field study at two locations to quantify larval density-dependent mortality in Bt and non-Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density. RESULTS: Increased pest density caused greater proportions of larvae to die in Bt maize than in non-Bt maize. All methods for calculating dose produced values less than high-dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did. CONCLUSION: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density-dependent mortality should be taken into account for both transgenic and non-transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry.
Subject(s)
Coleoptera , Endotoxins , Larva , Plants, Genetically Modified , Population Density , Zea mays , Zea mays/genetics , Animals , Coleoptera/drug effects , Coleoptera/growth & development , Coleoptera/genetics , Larva/growth & development , Larva/drug effects , Plants, Genetically Modified/genetics , Endotoxins/genetics , Endotoxins/pharmacology , Bacillus thuringiensis/genetics , Pest Control, Biological , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Hemolysin Proteins/pharmacology , Insecticide Resistance/geneticsABSTRACT
Genetically engineered (GE) cotton event MON 88702, producing Mpp51Aa2 (previously mCry51Aa2) from Bacillus thuringiensis (Bt), controls sucking pests, such as Lygus spp. (Hemiptera: Miridae) and thrips (Thysanoptera). Ingesting high doses of the insecticidal protein resulted in adverse effects on life table parameters of beneficial, predatory Orius spp. (Hemiptera: Anthocoridae). This triggered laboratory studies with more realistic food treatments, including different combinations of prey types with and without Bt protein to further characterize risks to this important group of non-target organisms. In this work, exclusive feeding of frozen spider mites (Tetranychus urticae, Acari: Tetranychidae) from Bt cotton confirmed adverse effects on longevity and fecundity of O. majusculus adults. Alternate feeding of Bt protein-containing spider mites and Bt-free Ephestia kuehniella (Lepidoptera: Pyralidae) eggs mitigated effects on longevity, but not on fecundity. When living larvae of Spodoptera littoralis (Lepidoptera: Noctuidae) from Bt cotton were fed to the predators, however, no effects on longevity and reproduction of female O. majusculus were observed, despite the fact that Bt protein concentrations in larvae were almost as high as concentrations in spider mites. When a diverse mix of prey species with various Bt protein concentrations is consumed in the field, it is unlikely that exposure of Orius spp. to Mpp51Aa2 is high enough to exert adverse effects on predator populations. MON 88702 cotton may thus be a valuable tool for integrated management of sucking pests.
Subject(s)
Bacillus thuringiensis , Gossypium , Longevity , Pest Control, Biological , Plants, Genetically Modified , Reproduction , Animals , Gossypium/genetics , Gossypium/parasitology , Gossypium/growth & development , Gossypium/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/parasitology , Bacillus thuringiensis/genetics , Reproduction/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Predatory Behavior , Fertility/genetics , Spodoptera/growth & development , Spodoptera/physiology , Spodoptera/genetics , Larva/growth & development , Larva/genetics , Bacillus thuringiensis Toxins/genetics , Endotoxins/genetics , Endotoxins/metabolism , Heteroptera/genetics , Heteroptera/physiology , Heteroptera/growth & development , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Tetranychidae/genetics , FemaleABSTRACT
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.
Subject(s)
Bacillus thuringiensis , Moths , Pesticides , Animals , Larva/genetics , Larva/metabolism , Glycine max/genetics , Endotoxins/genetics , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Pest Control, Biological/methods , Moths/metabolism , Bacillus thuringiensis/genetics , Bacillus thuringiensis/chemistry , Bacillus thuringiensis/metabolism , Chromosomes/metabolism , Hemolysin Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Insecticide Resistance/geneticsABSTRACT
The expression of Bacillus thuringiensis (Bt) toxins in transgenic cotton confers resistance to insect pests. However, it has been demonstrated that its effectiveness varies among cotton cultivars and different tissues. In this study, we evaluated the expression of Bt protein in 28 cotton cultivars and selected 7 cultivars that differed in Bt protein expression for transcriptome analysis. Based on their Bt protein expression levels, the selected cultivars were categorized into three groups: H (high Bt protein expression), M (moderate expression), and L (low expression). In total, 342, 318, and 965 differentially expressed genes were detected in the H vs. L, M vs. L, and H vs. M comparison groups, respectively. And three modules significantly associated with Bt protein expression were identified by weighted gene co-expression network analysis. Three hub genes were selected to verify their relationships with Bt protein expression using virus-induced gene silencing (VIGS). Silencing GhM_D11G1176, encoding an MYC transcription factor, was confirmed to significantly decrease the expression of Bt protein. The present findings contribute to an improved understanding of the mechanisms that influence Bt protein expression in transgenic cotton.