Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

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.

Development of a soybean leaf disc assay for determining oral insecticidal activity in the lepidopteran agricultural pest Helicoverpa armigera.

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.

Development of modified Cry1Ac for the control of resistant insect pest of cotton, Pectinophora gossypiella.

Cotton has been one of the most important cash crops in Pakistan, but its production is adversely affected by biotic and abiotic stresses. Insect pests such as pink bollworm present a colossal vulnerability to such a financially important commodity. Bt toxins have been widely used to safeguard agricultural plants against notorious insect pests such as cotton bollworm and pink bollworm, and they have proven to be effective in reducing chewing insect pests. However, its efficacy has been challenged due to the development of resistance in insect pests against Bt toxins such as Cry1Ac and this poses a significant risk to the long-term adoption of these Bt crops. Resistance in insect pests against Bt toxin Cry1Ac is developed due to the mutations in the midgut receptors such as cadherin. In this study first 56 amino acids which also includes helix alpha-1 portion from N-terminus of the Cry1Ac were removed and the gene was commercially synthesized following codon optimization. Modified Cry1Ac was used to develop transgenic plants of Nicotiana tabacum and insect bioassays were conducted to check the efficacy of Cry1Ac through leaf bioassays. Cry1Ac, a modified Bt toxin, was produced pET-28a (+), and diet bioassays were performed using purified protein at various doses against Pectinophora gossypiella. Based on the insect mortality and LC50, the Cry1AcM3 form of the modified toxins was shown to be more potent than the other modified versions (Cry1AcM1, Cry1AcM2), with more than 80 % mortality against resistant pink bollworm at 1.25 g/mL and an LC50 of 0.48. The results suggest that modified toxin cry1Ac may be useful in controlling population of pink bollworm resistant against cry1Ac.

MAPK-Activated Transcription Factor PxJun Suppresses PxABCB1 Expression and Confers Resistance to Bacillus thuringiensis Cry1Ac Toxin in Plutella xylostella (L.).

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that mitogen-activated protein kinase (MAPK)-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Here, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. IMPORTANCE The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system.

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.

Independent and Synergistic Effects of Knocking out Two ABC Transporter Genes on Resistance to Bacillus thuringiensis Toxins Cry1Ac and Cry1Fa in Diamondback Moth.

Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world's most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes.

Bacillus thuringiensis subsp. israelensis produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in Aedes aegypti larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.

The Cytocidal Spectrum of Bacillus thuringiensis Toxins: From Insects to Human Cancer Cells.

Bacillus thuringiensis (Bt) is a ubiquitous bacterium in soils, insect cadavers, phylloplane, water, and stored grain, that produces several proteins, each one toxic to different biological targets such as insects, nematodes, mites, protozoa, and mammalian cells. Most Bt toxins identify their particular target through the recognition of specific cell membrane receptors. Cry proteins are the best-known toxins from Bt and a great amount of research has been published. Cry are cytotoxic to insect larvae that affect important crops recognizing specific cell membrane receptors such as cadherin, aminopeptidase-N, and alkaline phosphatase. Furthermore, some Cry toxins such as Cry4A, Cry4B, and Cry11A act synergistically with Cyt toxins against dipteran larvae vectors of human disease. Research developed with Cry proteins revealed that these toxins also could kill human cancer cells through the interaction with specific receptors. Parasporins are a small group of patented toxins that may or may not have insecticidal activity. These proteins could kill a wide variety of mammalian cancer cells by recognizing specific membrane receptors, just like Cry toxins do. Surface layer proteins (SLP), unlike the other proteins produced by Bt, are also produced by most bacteria and archaebacteria. It was recently demonstrated that SLP produced by Bt could interact with membrane receptors of insect and human cancer cells to kill them. Cyt toxins have a structure that is mostly unrelated to Cry toxins; thereby, other mechanisms of action have been reported to them. These toxins affect mainly mosquitoes that are vectors of human diseases like Anopheles spp (malaria), Aedes spp (dengue, zika, and chikungunya), and Culex spp (Nile fever and Rift Valley fever), respectively. In addition to the Cry, Cyt, and parasporins toxins produced during spore formation as inclusion bodies, Bt strains also produce Vip (Vegetative insecticidal toxins) and Sip (Secreted insecticidal proteins) toxins with insecticidal activity during their vegetative growth phase.

Contribution of phenoloxidase activation mechanism to Bt insecticidal protein resistance in Asian corn borer.

Phenoloxidase (PO) is a crucial enzyme in the Arthropods melanization process, in which synthesized melanin rapidly acts at the site of injury and infection. In this study, we observed significant changes in humoral and cellular responses after exposing susceptible and resistant strains to a sub-lethal concentration of Cry1Ah toxin. Based on STRING v 11.0 computational protein-protein interaction analysis, we selected seven immune genes namely Prophenoloxidase PPO1b, PP03, Serpin-3, Serpin-5, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 reported in Asian corn borer. Quantitative real-time PCR gene expression studies showed Cry1Ah resistant strain had higher expression of PPO1b, PP03, Serpin-3, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 genes in midgut and hemocyte samples. This study also investigated and found that the level of prophenoloxidation (proPO) activity in Cry1Ah resistant strains was significantly higher than susceptible strains. Cry1Ah toxin significantly increased the resistant strain's immune responses, the difference was observed through assays of bacterial agglutination and phagocytosis. Additionally, immune response induced by Cry1Ah toxin influences the microbiome composition associated with the host system. These parameters seem to explain the contribution of PO/PO regulating proteins render the host to resist the Cry1Ah toxin.