Journal articles: 'Insecticidal protein'

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Jiang, Kun, Yan Zhang, Zhe Chen, Dalei Wu, Jun Cai, and Xiang Gao. "Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis." Toxins 12, no. 7 (July 5, 2020): 438. http://dx.doi.org/10.3390/toxins12070438.

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The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11200–789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides.

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Jiang, Kun, Xiaoyue Hou, Lu Han, Tongtong Tan, Zhanglei Cao, and Jun Cai. "Fibroblast Growth Factor Receptor, a Novel Receptor for Vegetative Insecticidal Protein Vip3Aa." Toxins 10, no. 12 (December 18, 2018): 546. http://dx.doi.org/10.3390/toxins10120546.

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Vegetative insecticidal proteins (Vips), which are secreted by some Bacillus thuringiensis strains during vegetative growth, exhibit high virulence to many pests. Vip3A proteins have been used commercially both in some bio-insecticides and in transgenic crops; however, compared with insecticidal crystal proteins, the mechanism of action of Vip3A is still unclear. In this work, we indicated that the fibroblast growth factor receptor-like protein (Sf-FGFR) from the membrane of Sf9 cells could bind to Vip3Aa. The interaction between Vip3Aa and Sf-FGFR was confirmed by pull-down assays and dot blotting experiment in vitro. The binding affinity between Vip3Aa and extracellular regions of Sf-FGFR (GST-FGFR-N) was determined by microscale thermophoresis assay (MST). Moreover, Vip3Aa-Flag could be co-immunoprecipitated with Sf-FGFR-V5 ex vivo. Furthermore, knockdown of Sf-FGFR gene in Sf9 cells resulted in reducing the mortality of those cells to Vip3Aa. In summary, our data indicated that Sf-FGFR is a novel receptor for Vip3Aa.

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Knox, Oliver G. G., Greg A. Constable, Bruce Pyke, and V. V. S. R. Gupta. "Environmental impact of conventional and Bt insecticidal cotton expressing one and two Cry genes in Australia." Australian Journal of Agricultural Research 57, no. 5 (2006): 501. http://dx.doi.org/10.1071/ar05366.

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Genetically modified Bt cotton, expressing the Cry1Ac protein for specific insecticidal activity against economically significant lepidopteran pests, has been available commercially in Australia since 1996. This technology has been improved and superseded by the addition of a second gene, allowing new varieties to express both the Cry1Ac the Cry2Ab proteins. Bt cotton offers several advantages to the grower, mainly through reduced insecticide spray requirements. The environmental benefits of reduced insecticide usage are assessed in this paper using the environmental impact quotient (EIQ). The assessment included consideration of the impact of the expressed transgenic proteins Cry1Ac and Cry2Ab. EIQ values of the Cry1Ac and Cry2Ab proteins were calculated at 9.9 and 7.9, respectively. Bt protein expression, plant biomass, insecticide application records, constituent of active ingredient, and insecticide EIQ values were used to produce an environmental impact (EI) value for insecticide use (kg a.i./ha) for conventional non-GM and single- and 2-gene Bt cotton for the 1997–98 to 2003–04 seasons. Inclusion of the Cry proteins in the assessment increased the EI values for Bt cotton by only 2%. The average insecticide EI value, for 2002–03 and 2003–04 seasons, for conventional cotton was 135 kg a.i./ha, whereas for the 2-gene Bt variety it was only 28 kg a.i./ha. Results of the EI evaluation indicate that, due to changes in insecticidal choice and reduction in usage, there was a reduction of >64% in EI from growing Bt cotton compared with conventional non-GM cotton in Australia.

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Shan, Yinxue, Minghui Jin, Swapan Chakrabarty, Bo Yang, Qi Li, Ying Cheng, Lei Zhang, and Yutao Xiao. "Sf-FGFR and Sf-SR-C Are Not the Receptors for Vip3Aa to Exert Insecticidal Toxicity in Spodoptera frugiperda." Insects 13, no. 6 (June 14, 2022): 547. http://dx.doi.org/10.3390/insects13060547.

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Vip3Aa is a novel insecticidal protein secreted by Bacillus thuringiensis (Bt) during its vegetative growth stages. It has high insecticidal activity against lepidopteran pests such as Spodoptera frugiperda, and has no cross-resistance with Cry insecticidal proteins. As a new type of insecticide, it plays an important role in controlling agricultural pests. However, the insecticidal mechanism of the Vip3Aa toxin, especially its definite receptors, have not been fully revealed. In this study, the previously reported Vip3Aa receptor genes Sf-FGFR and Sf-SR-C were knocked out separately using the CRISPR/Cas9 system. Bioassay results showed that the sensitivity of these two knockout strains to Vip3Aa were not significantly changed compared to that of the normal strain. The current results are not consistent with the previously reports that Sf-SR-C and Sf-FGFR were the receptors of Vip3Aa in vitro. This suggests that the Sf-SR-C and Sf-FGFR genes we tested may not be critical in the mode of action of Vip3Aa in vivo in Spodoptera frugiperda.

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Rajagopal, Raman, Naresh Arora, Swaminathan Sivakumar, Nagarjun G. V. Rao, Sharad A. Nimbalkar, and Raj K. Bhatnagar. "Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin." Biochemical Journal 419, no. 2 (March 27, 2009): 309–16. http://dx.doi.org/10.1042/bj20081152.

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The bacterium Bacillus thuringiensis produces ICPs (insecticidal crystal proteins) that are deposited in their spore mother cells. When susceptible lepidopteran larvae ingest these spore mother cells, the ICPs get solubilized in the alkaline gut environment. Of approx. 140 insecticidal proteins described thus far, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation as well as the principal ICP introduced into crops as transgene for agricultural crop protection. The 135 kDa Cry1Ac protein, upon ingestion by the insect, is processed successively at the N- and C-terminus by the insect midgut proteases to generate a 65 kDa bioactive core protein. The activated core protein interacts with specific receptors located at the midgut epithilium resulting in the lysis of cells and eventual death of the larvae. A laboratory-reared population of Helicoverpa armigera displayed 72-fold resistance to the B. thuringiensis insecticidal protein Cry1Ac. A careful zymogram analysis of Cry1Ac-resistant insects revealed an altered proteolytic profile. The altered protease profile resulted in improper processing of the insecticidal protein and as a consequence increased the LC50 concentrations of Cry1Ac. The 135 kDa protoxin-susceptible insect larval population processed the protein to the biologically active 65 kDa core protein, while the resistant insect larval population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides. N-terminal sequencing of these 95 and 68 kDa polypeptides produced by gut juices of resistant insects revealed an intact N-terminus. Protease gene transcription profiling by semi-quantitative RT (reverse transcription)–PCR led to the identification of a down-regulated HaSP2 (H. armigera serine protease 2) in the Cry1Ac-resistant population. Protease HaSP2 was cloned, expressed and demonstrated to be responsible for proper processing of insecticidal protoxin. The larval population displaying resistance to Cry1Ac do not show an altered sensitivity against another insecticidal protein, Cry2Ab. The implications of these observations in the context of the possibility of development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation are discussed.

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Lu, Xingxing, Huan Xu, Xiaoming Zhang, Tengda Sun, Yufan Lin, Yongheng Zhang, Honghong Li, et al. "Design, Synthesis and Bioactivity of Novel Low Bee-Toxicity Compounds Based on Flupyrimin." Molecules 27, no. 18 (September 19, 2022): 6133. http://dx.doi.org/10.3390/molecules27186133.

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Neonicotinoids are important insecticides for controlling aphids in agriculture. Growing research suggested that neonicotinoid insecticides are a key factor causing the decline of global pollinator insects, such as bees. Flupyrimin (FLP) is a novel nicotinic insecticide with unique biological properties and no cross-resistance, and is safe for pollinators. Using FLP as the lead compound, a series of novel compounds were designed and synthesized by replacing the amide fragment with a sulfonamideone. Their structures were confirmed by 1H NMR, 13C NMR and HRMS spectra. Bioassay results showed that compound 2j had good insecticidal activity against Aphis glycines with an LC50 value of 20.93 mg/L. Meanwhile, compound 2j showed significantly lower acute oral and contact toxicity to Apis mellifera. In addition, compound 2j interacted well with the protein in insect acetylcholine binding protein (AChBP). The molecular docking on honeybee nicotinic acetylcholine receptor (nAChR) indicated that the sulfonamide group of compound 2j did not form a hydrogen bond with Arg173 of the β subunit, which conforms to the reported low bee-toxicity conformation. In general, target compound 2j can be regarded as a bee-friendly insecticide candidate.

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Ellis, R. Tracy, Brian A. Stockhoff, Lisa Stamp, H. Ernest Schnepf, George E. Schwab, Mark Knuth, Josh Russell, Guy A. Cardineau, and Kenneth E. Narva. "Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte." Applied and Environmental Microbiology 68, no. 3 (March 2002): 1137–45. http://dx.doi.org/10.1128/aem.68.3.1137-1145.2002.

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ABSTRACT A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.

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Sopko, Megan S., Kenneth E. Narva, Andrew J. Bowling, Heather E. Pence, James J. Hasler, Theodore J. Letherer, Cory M. Larsen, and Marc D. Zack. "Modification of Vip3Ab1 C-Terminus Confers Broadened Plant Protection from Lepidopteran Pests." Toxins 11, no. 6 (June 3, 2019): 316. http://dx.doi.org/10.3390/toxins11060316.

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Vegetative insecticidal proteins (Vips) from Bacillus thuringiensis (Bt) are unique from crystal (Cry) proteins found in Bt parasporal inclusions as they are secreted during the bacterial vegetative growth phase and bind unique receptors to exert their insecticidal effects. We previously demonstrated that large modifications of the Vip3 C-terminus could redirect insecticidal spectrum but results in an unstable protein with no lethal activity. In the present work, we have generated a new Vip3 protein, Vip3Ab1-740, via modest modification of the Vip3Ab1 C-terminus. Vip3Ab1-740 is readily processed by midgut fluid enzymes and has lethal activity towards Spodoptera eridania, which is not observed with the Vip3Ab1 parent protein. Importantly, Vip3Ab1-740 does retain the lethal activity of Vip3Ab1 against other important lepidopteran pests. Furthermore, transgenic plants expressing Vip3Ab1-740 are protected against S. eridania, Spodoptera frugiperda, Helicoverpa zea, and Pseudoplusia includens. Thus, these studies demonstrate successful engineering of Vip3 proteins at the C-terminus to broaden insecticidal spectrum, which can be employed for functional expression in planta.

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López-Pazos, S. A., F. M. Chavarrio Cañas, and A. C. Rojas Arias. "Insecticidal and Potato Growth Stimulation Activity of Bacillus thuringiensis kurstaki HD-1." Mikrobiolohichnyi Zhurnal 84, no. 4 (January 17, 2023): 9–29. http://dx.doi.org/10.15407/microbiolj84.04.009.

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Bacillus thuringiensis (Bt) produces Cry toxins against pest insects. Cry proteins are conformed by domains related to pore formation and recognition of protein receptors. Plant-induced systemic resistance (ISR) is triggered due to pest attack, it could be activated by Bacillus sp. Tecia solanivora (Ts) is a potato pest, susceptible to Cry1Ac and Cry1B proteins. This paper indicates the endorsement of Bt kurstaki HD-1 (BtkHD1) in relation to Ts control (Cry1Ac and Cry1B proteins), potato growth promotion, and plant ISR due to pests related to the BtkHD1-potato system. To ensure that ongoing quality control of BtkHD1 was maintained, crystal synthesis (microscopy), cry1 genes presence, and Cry protein production were checked. Bioassays Ts larvae and potato plantlets and an in silico analysis of the hybrid Cry1Ac-Cry1Ba protein and potato ISR related to the BtkHD1 infl uence were performed. Bioassay on Ts larvae shows an LC50 of 536 ng/cm2 of diet. A potato growth promotion assay revealed the effect of BtkHD1 on the length and dry weight of stems. The prospective analysis took into account relevant factors affecting the biological function of the hybrid protein focused on domain II. In silico identification of 15 BtkHD1 proteins and 68 potato proteins related to plant ISR due to pests was completed. This project serves to validation of toxicity on Ts larvae and potato growth effect based on BtkHD1, including a forward analysis of the hybrid Cry1Ac1-Cry1Ba1, and proteins associated with this strain and potato for eliciting plant ISR due to pests.

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Edrington, Thomas, Rong Wang, Lucas McKinnon, Colton Kessenich, Kimberly Hodge-Bell, Wenze Li, Jianguo Tan, et al. "Food and feed safety of the Bacillus thuringiensis derived protein Vpb4Da2, a novel protein for control of western corn rootworm." PLOS ONE 17, no. 8 (August 3, 2022): e0272311. http://dx.doi.org/10.1371/journal.pone.0272311.

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Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals.

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Shim, Hee Jin, Jae Young Choi, Yong Wang, Xue Ying Tao, Qin Liu, Jong Yul Roh, Jae Su Kim, et al. "NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide." Applied and Environmental Microbiology 79, no. 1 (October 12, 2012): 141–49. http://dx.doi.org/10.1128/aem.02781-12.

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ABSTRACTA novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, theBacillus thuringiensiscrystal protein gene (here termedcry1-5) was introduced into theAutographa californicanucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene,AaIT, fromAndroctonus australiswas introduced under the control of an early promoter fromCotesia plutellaebracovirus by fusion of a partial fragment oforf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity againstPlutella xylostellalarvae and a significant reduction in the median lethal time againstSpodoptera exigualarvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in whichAaITand/orcry1-5were deleted were generated by serial passagesin vitro. Expression of the foreign proteins (B. thuringiensistoxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected fromS. exigualarvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

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Pan, Zhi-Zhen, Lian Xu, Yi-Shu Zheng, Li-Yang Niu, Bo Liu, Nan-Yan Fu, Yan Shi, Qing-Xi Chen, Yu-Jing Zhu, and Xiong Guan. "Synthesis and Characterization of Cry2Ab–AVM Bioconjugate: Enhanced Affinity to Binding Proteins and Insecticidal Activity." Toxins 11, no. 9 (August 27, 2019): 497. http://dx.doi.org/10.3390/toxins11090497.

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Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4”-O-succinyl avermectin (AVM) to synthesize Cry2Ab–AVM bioconjugate. It was found that Cry2Ab–AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab–AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10–11), with a much higher affinity (dissociation equilibrium constant KD = 3.44 nM) than Cry2Ab30 (KD = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab–AVM ligand docking into the PxCR10–11 is a potential mechanism to enhance the binding affinity of Cry2Ab–AVM to PxCR10–11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.

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Gomis-Cebolla, Joaquín, Iñigo Ruiz de Escudero, Natalia Mara Vera-Velasco, Patricia Hernández-Martínez, Carmen Sara Hernández-Rodríguez, Tomás Ceballos, Leopoldo Palma, Baltasar Escriche, Primitivo Caballero, and Juan Ferré. "Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein." Journal of Invertebrate Pathology 142 (January 2017): 60–67. http://dx.doi.org/10.1016/j.jip.2016.10.001.

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IIZUKA, Toshihiko. "Insecticidal crystal protein and its agricultural utilization." Kagaku To Seibutsu 27, no. 5 (1989): 287–96. http://dx.doi.org/10.1271/kagakutoseibutsu1962.27.287.

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Naimov, Samir, Rumyana Boncheva, Rumyana Karlova, Stefan Dukiandjiev, Ivan Minkov, and Ruud A. de Maagd. "Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins." Applied and Environmental Microbiology 74, no. 23 (October 3, 2008): 7145–51. http://dx.doi.org/10.1128/aem.00752-08.

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ABSTRACT Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

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Stock, Carolyn A., Thomas J. McLoughlin, Janet A. Klein, and Michael J. Adang. "Expression of a Bacillus thuringiensis crystal protein gene in Pseudomonas cepacia 526." Canadian Journal of Microbiology 36, no. 12 (December 1, 1990): 879–84. http://dx.doi.org/10.1139/m90-152.

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An insecticidal crystal protein gene, cryIA(C), from Bacillus thuringiensis HD-1 was cloned into a broad host range vector, pSUP204, and the resulting plasmid, pSUP89A, was conjugated into a plant-colonizing bacterium, Pseudomonas cepacia 526. Southern blot analysis detected the presence of the cry gene in Pseudomonas cepacia 526 transconjugants. Production of a truncated insecticidal crystal protein in Pseudomonas cepacia was detected by Western blotting and ELISA techniques and was found to have insecticidal activity against the tobacco hornworm, Manduca sexta, in an artificial diet assay. We also followed the establishment of the Pseudomonas cepacia 526/Bt transconjugant on axenically grown tobacco plants and found that approximately 1% of the Pseudomonas cells retained pSUP89A 3 days after application. In spite of this instability, treated tobacco plants were protected from Manduca sexta infestation. Key words: Bacillus thuringiensis, Pseudomonas cepacia, insecticidal crystal protein, Cry protein.

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Selvapandiyan, A., N. Arora, R. Rajagopal, S. K. Jalali, T. Venkatesan, S. P. Singh, and Raj K. Bhatnagar. "Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis." Applied and Environmental Microbiology 67, no. 12 (December 1, 2001): 5855–58. http://dx.doi.org/10.1128/aem.67.12.5855-5858.2001.

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ABSTRACT A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.

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Kerns, Dawson D., David L. Kerns, Gus M. Lorenz, Angus L. Catchot Jr., and Scott D. Stewart. "Impact of Various Bt Cotton Traits and the Application of an Insecticide on the Within Plant Distribution of Helicoverpa zea (Lepidoptera: Noctuidae) Larvae and Injured Floral Structures." Journal of Cotton Science 24, no. 4 (2020): 148–58. http://dx.doi.org/10.56454/huoe5710.

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Previous studies have indicated that the expression of insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) in cotton can have a significant influence on the behavior of bollworm larvae (Helicoverpa zea, Lepidoptera:Noctuidae). This suggests that the particular Bt protein produced by a cotton variety may need to be considered when determining the most ideal scouting methods to utilize for bollworm. NonBt, WideStrike (producing Cry1Ac + Cry1F Bt insecticidal proteins), and Bollgard II (Cry1Ac + Cry2Ab) cotton varieties were planted and either treated with an insecticide or left untreated. The presence of H. zea larvae and their feeding injury were recorded according to their location in the canopy and type of floral structure where they were found. Results from comparison of larval and injury distributions indicated no significant differences between the different cotton varieties tested, and that insecticide treatment had minimal impact on this distribution. Larval size was generally associated with location in the canopy, suggesting that larvae tend to move towards the middle of the canopy as they age. The effect of different Bt cotton technologies appears to associate with how quickly larvae move to preferred feeding sites rather than their preference for particular feeding sites. These results suggest that scouting methods could be standardized independently of the presence of a Bt cotton trait or previous insecticide application. Focusing scouting efforts on the middle portion of the canopy (i.e., nodes 6-9) should increase the detection of small larvae and ‘fresh’ injury and be less influenced by previous insecticide applications.

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Kouadio, Jean-Louis, Meiying Zheng, Michael Aikins, David Duda, Stephen Duff, Danqi Chen, Jun Zhang, et al. "Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm." PLOS ONE 16, no. 12 (December 20, 2021): e0260532. http://dx.doi.org/10.1371/journal.pone.0260532.

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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel β-sheets organized into β-sandwich layers. The amino-terminal domains 1–3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long β-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4–6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

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Walters, Frederick S., Scott Young, and Gerson Graser. "Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098." Transgenic Research 29, no. 1 (November 28, 2019): 109–24. http://dx.doi.org/10.1007/s11248-019-00183-w.

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AbstractSafety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.

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Nishiwaki, Hisashi, Kenta Nakashima, Chiharu Ishida, Tadayuki Kawamura, and Kazuhiko Matsuda. "Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by Bacillus sphaericus." Applied and Environmental Microbiology 73, no. 10 (March 30, 2007): 3404–11. http://dx.doi.org/10.1128/aem.00021-07.

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ABSTRACT An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.

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Sun, Hong, Yifan Li, Xinyu Li, and Yalin Zhang. "The Inhibition of Serine/Threonine Protein Phosphatase Type 5 Mediates Cantharidin Toxicity to Control Periplaneta americana (L.)." Insects 11, no. 10 (October 8, 2020): 682. http://dx.doi.org/10.3390/insects11100682.

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The American cockroach, Periplaneta americana (L.), is a notorious urban pest. It has developed insecticidal resistance to commonly used insecticides. Cantharidin (CTD) is a defensive toxin derived from blister beetles. It has been verified to have insecticidal toxicity in a range of pests. In this study, we determined the ingestion toxicity of CTD and norcantharidin (NCTD) to P. americana to test whether they had the potential to be effective against P. americana. Bioassays revealed that CTD produces toxicity against P. americana. The median lethal concentration (LC50) value of CTD was 50.92 μg/mL, while NCTD displayed nearly no toxicity against P. americana. The inhibition assays of serine/threonine protein phosphatases (PSPs) in P. americana indicated that CTD and NCTD could inhibit PSPs. The value of the half maximal inhibitory concentration (IC50) of CTD was 7.21 ± 0.94 μM, whereas that of NCTD was higher, at 31.65 ± 3.87 μM. Furthermore, the inhibition effect of CTD on the serine/threonine protein phosphatase type 5 of P. americana (PaPP5) was superior to that of NCTD. Specifically, the IC50 of CTD reached 0.39 ± 0.04 μM, while the IC50 of NCTD was 1.87 ± 0.23 μM. This study paves the way for insect-derived agents (CTD) to be applied toward controlling P. americana and contributes to the development of novel insecticides based on PP5 as a target.

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Morgan, J. Alun W., Martin Sergeant, Debbie Ellis, Margaret Ousley, and Paul Jarrett. "Sequence Analysis of Insecticidal Genes fromXenorhabdus nematophilus PMFI296." Applied and Environmental Microbiology 67, no. 5 (May 1, 2001): 2062–69. http://dx.doi.org/10.1128/aem.67.5.2062-2069.2001.

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ABSTRACT Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared inEscherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC50] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1gene from the bacteriophage lambda P L promoter were shown to have insecticidal activity (LC50 of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilusPMFI296.

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Dahal, Sagar, Jaishree Sijapati, and Sandeep Rijal. "Screening and Confirmation of Vip3 gene presence in Bacillus thuringiensis strains native to Nepal." International Journal of Applied Sciences and Biotechnology 10, no. 1 (March 29, 2022): 71–74. http://dx.doi.org/10.3126/ijasbt.v10i1.44162.

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An insecticidal protein named Vegetative Insecticidal Protein (Vip) produced by Bacillus thuringiensis during its vegetative growth is gaining its popularity as second-generation insecticidal protein because of its lethal effect on insects of class Coleoptera, Lepidoptera, Diptera. The research was designed for the confirmation of Vip3 gene presence in soil samples collected from different geographical region of Nepal. For the purpose, 36 soil samples from different places of Nepal were collected. Primers were used for PCR amplification of DNA templates extracted and product size was observed around 1621bp.Out of total sample analyzed, 10% of sample showed the presence of Vip3 gene. Int. J. Appl. Sci. Biotechnol. Vol 10(1): 71-74.

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Bakhsh, A., K. Shahzad, and T. Husnain. "Variation in the spatio-temporal expression of insecticidal genes in cotton." Czech Journal of Genetics and Plant Breeding 47, No. 1 (March 18, 2011): 1–9. http://dx.doi.org/10.17221/131/2010-cjgpb.

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The most significant breakthrough in plant biotechnology is the development of the techniques to transform genes from unrelated sources into commercially important crop plants to develop resistance against targeted insect pests. The spatio-temporal expression of insecticidal genes in transgenic cotton varies with plant age, plant parts and environmental conditions. The understanding of this temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production. This review summarizes variations in the efficacy of introduced insecticidal genes in cotton crop. The factors contributing to the variability of endotoxins have also been highlighted. The reduction in Bt protein biosynthesis in late-season cotton tissues could be attributed to the overexpression of the Bt gene at earlier stages, which leads to gene regulation at post-transcription levels and consequently results in gene silencing at a later stage. Methylation of the promoter may also play a role in the declined expression of endotoxin proteins. In genetically modified crops several environmental factors have been reported to affect the expression of transgenes. Among environmental factors nitrogen metabolism, inhibition of synthesis, degradation, remobilization and high temperature are attributable to the quantitative reduction in Bt proteins. Applying plant growth regulators or protein enhancers such as Chaperone<sup>TM</sup> may improve Bt cotton efficacy through enhancing the synthesis of proteins. Also some agronomic practices such as nitrogen fertilization and timely irrigation favour the endotoxin expression. Thus, variations in the efficacy of insecticidal genes in transgenic cotton and the involved mechanisms need to be understood fully so as to plan rational resistance management strategies to retard the rate of resistance development and to control target pests effectively by enhancing the endotoxin expression through genetic or agronomic management.

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Barreto, Marliton R., Leandro L. Loguercio, Fernando H. Valicente, and Edilson Paiva. "Insecticidal activity of culture supernatants from Bacillus thuringiensis Berliner strains against Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) larvae." Anais da Sociedade Entomológica do Brasil 28, no. 4 (December 1999): 675–85. http://dx.doi.org/10.1590/s0301-80591999000400010.

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Novel vegetative insecticidal proteins (Vips) identified in the supernatant of Bacillus thuringiensis (B.t.) cultures have shown to provide adequate control over a wide spectrum of economically important crop pests. To evaluate the potential applicability of these proteins against fall armyworm (Spodoptera frugiperda Smith) larvae, the most important insect pest for tropical maize, the characteristics and mortality effects of culture supernatants from five B.t. strains were investigated. Striking differences among strains were detected, not only in terms of efficiency in killing the insect, but also regarding to mortality effects of heated and non-heated supernatants, which were used to distinguish the heat-sensitive protein-derived insecticidal fraction from a thermostable one, with a non-protein nature (b-exotoxinas). The qualitative, quantitative and temporal patterns of total protein secretion in the medium (supernatant) were assessed through spectrophotometry and polyacrylamide gel electrophoresis. The strains showed remarkably distinct rates of growth and timing for protein secretion relative to cell density in culture. Moreover, the electrophoretic-banding patterns also varied in a strain-specific manner, both in denaturing and non denaturing conditions. Polypeptides displaying a molecular weight that is very close to the expected for previously identified Vip3A proteins were found for the strains with high supernatant-mortality ratios. The data suggest the feasibility and usefulness of searching for protein-derived (Vip-like) insecticidal fractions in B.t. supernatants as a mean of developing especific and efficient alternatives of biological control to be employed in integrated pest management programs of S. frugiperda in tropical maize.

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Dahmana, Handi, Didier Raoult, Florence Fenollar, and Oleg Mediannikov. "Insecticidal Activity of Bacteria from Larvae Breeding Site with Natural Larvae Mortality: Screening of Separated Supernatant and Pellet Fractions." Pathogens 9, no. 6 (June 18, 2020): 486. http://dx.doi.org/10.3390/pathogens9060486.

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Mosquitoes can transmit to humans devastating and deadly pathogens. As many chemical insecticides are banned due to environmental side effects or are of reduced efficacy due to resistance, biological control, including the use of bacterial strains with insecticidal activity, is of increasing interest and importance. The urgent actual need relies on the discovery of new compounds, preferably of a biological nature. Here, we explored the phenomenon of natural larvae mortality in larval breeding sites to identify potential novel compounds that may be used in biological control. From there, we isolated 14 bacterial strains of the phylum Firmicutes, most of the order Bacillales. Cultures were carried out under controlled conditions and were separated on supernatant and pellet fractions. The two fractions and a 1:1 mixture of the two fractions were tested on L3 and early L4 Aedes albopictus. Two concentrations were tested (2 and 6 mg/L). Larvae mortality was recorded at 24, 48 and 72 h and compared to that induced by the commercialized B. thuringiensis subsp. israelensis. Of the 14 strains isolated, 11 were active against the A. albopictus larvae: 10 of the supernatant fractions and one pellet fraction, and mortality increased with the concentration. For the insecticide activity prediction in three strains of the Bacillus cereus complex, PCR screening of the crystal (Cry) and cytolytic (Cyt) protein families characteristic to B. thuringiensis subsp. israelensis was performed. Most of the genes coding for these proteins’ synthesis were not detected. We identified bacterial strains that exhibit higher insecticidal activity compared with a commercial product. Further studies are needed for the characterization of active compounds.

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Vazquez-Padron, Roberto I., Gustavo de la Riva, Guillermin Agüero, Yussun Silva, Si M. Pham, Mario Soberón, Alejandra Bravo, and Abdelouahab Aı̈touche. "Cryptic endotoxic nature ofBacillus thuringiensisCry1Ab insecticidal crystal protein." FEBS Letters 570, no. 1-3 (June 19, 2004): 30–36. http://dx.doi.org/10.1016/j.febslet.2004.06.021.

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Corbin, David R., Frederick J. Perlak, David A. Fischhoff, John T. Greenplate, Zhen Shen, and John P. Purcell. "New Proteins for the Control of Insects in Transgenic Crops." HortScience 31, no. 4 (August 1996): 699b—699. http://dx.doi.org/10.21273/hortsci.31.4.699b.

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Genetically modified potato and cotton crops that express insecticidal proteins from Bacillus thuringiensis (Bt) have recently been commercialized. These crops display autonomous resistance to specific insect pests, and thus offer major agricultural and environmental benefits. We have implemented a microbial screening program to discover new types of insecticidal proteins for use in transgenic crops. New proteins with diverse modes of action offer opportunities to control insect pests that are not susceptible to Bt insecticidal proteins and to delay or prevent the potential occurrence of resistance of insects to crops genetically modified with Bt genes. Cholesterol oxidase emerged from our screen as a new insecticidal protein with potent activity against the cotton boll weevil. Cholesterol oxidase was acutely toxic to boll weevil larvae, with an LC50 of 2–6 parts per million when ingested in artificial diet feeding assays, and caused marked reductions in fecundity when ingested by adult boll weevils. Cholesterol oxidase also exerted significant, though less severe, toxicity against several lepidopteran pests. The insecticidal action of cholesterol oxidase appears to be due to oxidation of midgut epithelial membrane cholesterol followed by membrane disruption. A cholesterol oxidase gene was cloned and expressed in transgenic tobacco plants to yield plant tissue that exerted potent activity against boll weevil. Expression of this cholesterol oxidase gene in cotton plants may offer significant protection against the cotton boll weevil and may also aid in the mitigation of resistance of cotton lepidopteran pests to Bt proteins.

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Peña, Guadalupe, Juan Miranda-Rios, Gustavo de la Riva, Liliana Pardo-López, Mario Soberón, and Alejandra Bravo. "A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)." Applied and Environmental Microbiology 72, no. 1 (January 2006): 353–60. http://dx.doi.org/10.1128/aem.72.1.353-360.2006.

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ABSTRACT The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.

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Rathinam, Maniraj, Karthik Kesiraju, Shweta Singh, Vinutha Thimmegowda, Vandna Rai, Debasis Pattanayak, and Rohini Sreevathsa. "Molecular Interaction-Based Exploration of the Broad Spectrum Efficacy of a Bacillus thuringiensis Insecticidal Chimeric Protein, Cry1AcF." Toxins 11, no. 3 (March 2, 2019): 143. http://dx.doi.org/10.3390/toxins11030143.

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Bacillus thuringiensis insecticidal proteins (Bt ICPs) are reliable and valuable options for pest management in crops. Protein engineering of Bt ICPs is a competitive alternative for resistance management in insects. The primary focus of the study was to reiterate the translational utility of a protein-engineered chimeric Cry toxin, Cry1AcF, for its broad spectrum insecticidal efficacy using molecular modeling and docking studies. In-depth bioinformatic analysis was undertaken for structure prediction of the Cry toxin as the ligand and aminopeptidase1 receptors (APN1) from Helicoverpa armigera (HaAPN1) and Spodoptera litura (SlAPN1) as receptors, followed by interaction studies using protein-protein docking tools. The study revealed feasible interactions between the toxin and the two receptors through H-bonding and hydrophobic interactions. Further, molecular dynamics simulations substantiated the stability of the interactions, proving the broad spectrum efficacy of Cry1AcF in controlling H. armigera and S. litura. These findings justify the utility of protein-engineered toxins in pest management.

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Vieira, Tatiana F., Maria F. Araújo, Maria José G. Fernandes, David M. Pereira, A. Gil Fortes, Elisabete M. S. Castanheira, M. Sameiro T. Gonçalves, and Sérgio F. Sousa. "In Silico Identification of Protein Targets Associated to the Insecticide Activity of Eugenol Derivatives." Chemistry Proceedings 3, no. 1 (November 14, 2020): 138. http://dx.doi.org/10.3390/ecsoc-24-08333.

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The control of insect pests and the need for increased food production due to the world population growth, together with environmental issues associated with synthetic pesticides, has stimulated the development of new and “greener” alternatives, based on natural compounds. Eugenol is a natural compound that is the major component of clove oil. It has demonstrated antimicrobial and antioxidant activity, being also a powerful insecticide. Recently, new eugenol derivatives have been developed, with some molecules displaying increased insecticide activity. One of the difficulties associated with the rational development of new eugenol derivatives with enhanced insecticidal activity lies in the lack of knowledge of the specific protein target responsible for its activity and to the binding conformation of these molecules. Here, we report the application of an integrated molecular modeling—inverted virtual screening protocol of a collection of eugenol derivatives with confirmed insecticide activity against a molecular library of protein targets typically associated with the insecticide activity of natural compounds. The protocol included six different scoring functions from popular docking software alternatives. The results consistently show a marked preference for interaction of the eugenol derivatives with the odorant binding proteins (OBPs) in insect species. Interestingly, OBPs have been regarded as promising targets in the insect periphery nerve system for environmental-friendly approaches in insect pest management. The present results provide clues for the rational development of new eugenol derivatives as bioinsecticides targeting OBPs.

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Wang, Cunxi, Gregory J. Bean, Chun Ju Chen, Colton R. Kessenich, Jiexin Peng, Nicolo R. Visconti, Jason S. Milligan, et al. "Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm." PLOS ONE 17, no. 9 (September 8, 2022): e0274204. http://dx.doi.org/10.1371/journal.pone.0274204.

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The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (β-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

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Godbold, Russell, Whitney D. Crow, Jeffrey Gore, Fred Musser, Angus L. Catchot, Darrin M. Dodds, Don R. Cook, Tyler Towles, and Nathan S. Little. "Efficacy of Bt Toxins and Foliar Insecticides Against Bollworm, Helicoverpa zea (Boddie), in Dried Flower Corollas of Cotton." Journal of Cotton Science 27, no. 1 (2023): 28–36. http://dx.doi.org/10.56454/znax3626.

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Foliar insecticides and insecticidal protein from Bacillus thuringiensis (Bt) in transgenic cotton are common tools used for bollworm management in cotton. Efficacy of Bt proteins and foliar insecticides can be dependent upon larval location in the plant canopy and time of year. Floral structures are known to be a common food source for bollworm. Floral components can complicate bollworm control with foliar insecticides and transgenic cotton by protecting larvae from contact with formulated insecticides and lower concentrations of Bt proteins. Mortality was measured to evaluate the effects of Bt expression and foliar insecticides in flowers of non-Bt, two-gene Bt, and three-gene Bt cotton varieties. Expression of Bt protein in white flowers provided some efficacy after three days. Bollworm mortality at three days after infestation was less than 50% for all varieties in an unsprayed environment. Larval mortality on Bollgard 3 flowers was higher than that on Bollgard II flowers. Surviving larvae feeding on Bollgard 3 flowers weighed less than larvae that fed on Bollgard II flowers, and larvae that fed on Bollgard II flowers weighed less than those that fed on non-Bt flowers. The use of chlorantraniliprole or methoxyfenozide + spinetoram provided some control of bollworms three days after application to wilting flower corollas (bloom tags). Mortality ranged from 41.9% following application of chlorantraniliprole to 61.0% following application of methoxyfenozide + spinetoram. Results from this study will be used to improve integrated pest management programs for bollworm management in cotton.

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Miyasono, Minoru, Shyuichiro Inagaki, Reiji Tanaka, Yoshito Ueyama, and Reiji Takeda. "Enhancement of Insecticidal Protein Activity by Spores of Bacillus thuringiensis against the Diamondback Moth, Plutella xylostella, Developing Resistance to Insecticidal Protein." Japanese Journal of Applied Entomology and Zoology 47, no. 2 (2003): 61–66. http://dx.doi.org/10.1303/jjaez.2003.61.

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Yamashita, Satoko, Tetsuyuki Akao, Eiichi Mizuki, Hiroyuki Saitoh, Kazuhiko Higuchi, Yu Shin Park, Ho-San Kim, and Michio Ohba. "Characterization of the anti-cancer-cell parasporal proteins of aBacillus thuringiensisisolate." Canadian Journal of Microbiology 46, no. 10 (October 1, 2000): 913–19. http://dx.doi.org/10.1139/w00-084.

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An unusual activity, associated with non-insecticidal and non-haemolytic parasporal inclusion proteins of a Bacillus thuringiensis soil isolate, designated 89-T-26-17, was characterized. The parasporal inclusion of this isolate was bipyramidal, rounded at both ends, containing proteins of 180, 150, 120, 100, and 88 kDa. No homologies with the Cry and Cyt proteins of B. thuringiensis were detected based on N-terminal sequences. Proteolytic processing of the inclusion proteins by proteinase K, trypsin, and chymotrypsin produced a major protein of 64 kDa exhibiting cytocidal activity against human leukaemic T cells and uterus cervix cancer (HeLa) cells. The protease-activated proteins showed no cytotoxicity to normal T cells.Key words: Bacillus thuringiensis parasporal inclusion, non-insecticidal, non-haemolytic, cytocidal activity, human cancer cell.

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Yamagiwa, Masashi, Motoyuki Esaki, Kanao Otake, Manabu Inagaki, Tohru Komano, Teruo Amachi, and Hiroshi Sakai. "Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensissubsp. israelensis." Applied and Environmental Microbiology 65, no. 8 (August 1, 1999): 3464–69. http://dx.doi.org/10.1128/aem.65.8.3464-3469.1999.

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ABSTRACT Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp.israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathioneS-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.

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Martin, Macarena, Debora Boaventura, and Ralf Nauen. "Evaluation of Reference Genes and Expression Level of Genes Potentially Involved in the Mode of Action of Cry1Ac and Cry1F in a Susceptible Reference Strain of Chrysodeixis includens." Insects 12, no. 7 (June 30, 2021): 598. http://dx.doi.org/10.3390/insects12070598.

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Soybean looper (SBL), Chrysodeixis includens (Walker), is one of the major lepidopteran pests of soybean in the American continent. SBL control relies mostly on the use of insecticides and genetically modified crops expressing Bacillus thuringiensis (Bt) insecticidal Cry proteins. Due to the high selection pressure exerted by these control measures, resistance has developed to different insecticides and Bt proteins. Nevertheless, studies on the mechanistic background are still scarce. Here, the susceptibility of the laboratory SBL-Benzon strain to the Bt proteins Cry1Ac and Cry1F was determined in diet overlay assays and revealed a greater activity of Cry1Ac than Cry1F, thus confirming results obtained for other sensitive SBL strains. A reference gene study across larval stages with four candidate genes revealed that RPL10 and EF1 were the most stable genes for normalization of gene expression data obtained by RT-qPCR. Finally, the basal expression levels of eight potential Bt protein receptor genes in six larval instars were analyzed, including ATP-binding cassette (ABC) transporters, alkaline phosphatase, aminopeptidases, and cadherin. The results presented here provide fundamental knowledge to support future SBL resistance studies.

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Singh, Gatikrushna, Bindiya Sachdev, Nathilal Sharma, Rakesh Seth, and Raj K. Bhatnagar. "Interaction of Bacillus thuringiensis Vegetative Insecticidal Protein with Ribosomal S2 Protein Triggers Larvicidal Activity in Spodoptera frugiperda." Applied and Environmental Microbiology 76, no. 21 (September 10, 2010): 7202–9. http://dx.doi.org/10.1128/aem.01552-10.

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ABSTRACT Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

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Brown, S. E., A. T. Cao, P. Dobson, E. R. Hines, R. J. Akhurst, and P. D. East. "Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria." Applied and Environmental Microbiology 72, no. 2 (February 2006): 1653–62. http://dx.doi.org/10.1128/aem.72.2.1653-1662.2006.

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ABSTRACT Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

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Baum, James A., and Thomas Malvar. "Regulation of insecticidal crystal protein production in Bacillus thuringiensis." Molecular Microbiology 18, no. 1 (October 1995): 1–12. http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_18010001.x.

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Wei, Jun-Zhi, Jessica O'Rear, Ute Schellenberger, Barbara A. Rosen, Young-Jun Park, Mark J. McDonald, Genhai Zhu, et al. "A selective insecticidal protein fromPseudomonas mosseliifor corn rootworm control." Plant Biotechnology Journal 16, no. 2 (October 1, 2017): 649–59. http://dx.doi.org/10.1111/pbi.12806.

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Shan, Yueming, Changlong Shu, Kanglai He, Xue Cheng, Lili Geng, Wensheng Xiang, and Jie Zhang. "Characterization of a Novel Insecticidal Protein Cry9Cb1 fromBacillus thuringiensis." Journal of Agricultural and Food Chemistry 67, no. 13 (March 13, 2019): 3781–88. http://dx.doi.org/10.1021/acs.jafc.9b00385.

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OSBORNI, T. C., D. C. ALEXANDER, S. S. M. SUN, C. CARDONA, and F. A. BLISS. "Insecticidal Activity and Lectin Homology of Arcelin Seed Protein." Science 240, no. 4849 (April 8, 1988): 207–10. http://dx.doi.org/10.1126/science.240.4849.207.

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Pan, Xiaohong, Zhangyan Xu, Yilin Zheng, Tengzhou Huang, Lan Li, Zhi Chen, Wenhua Rao, Saili Chen, Xianxian Hong, and Xiong Guan. "The adsorption features between insecticidal crystal protein and nano-Mg(OH) 2." Royal Society Open Science 4, no. 12 (December 2017): 170883. http://dx.doi.org/10.1098/rsos.170883.

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Nano-Mg(OH) 2 , with low biological toxicity, is an ideal nano-carrier for insecticidal protein to improve the bioactivity. In this work, the adsorption features of insecticidal protein by nano-Mg(OH) 2 have been studied. The adsorption capacity could reach as high as 136 mg g −1 , and the adsorption isotherm had been fitted with Langmuir and Freundlich models. Moreover, the adsorption kinetics followed a pseudo-first or -second order rate model, and the adsorption was spontaneous and an exothermic process. However, high temperatures are not suitable for adsorption, which implies that the temperature would be a critical factor during the adsorption process. In addition, FT-IR confirmed that the protein was adsorbed on the nano-Mg(OH) 2 , zeta potential analysis suggested that insecticidal protein was loaded onto the nano-Mg(OH) 2 not by electrostatic adsorption but maybe by intermolecular forces, and circular dichroism spectroscopy of Cry11Aa protein before and after loading with nano-Mg(OH) 2 was changed. The study applied the adsorption information between Cry11Aa and nano-Mg(OH) 2 , which would be useful in the practical application of nano-Mg(OH) 2 as a nano-carrier.

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Fang, Jun, Xiaoli Xu, Ping Wang, Jian-Zhou Zhao, Anthony M. Shelton, Jiaan Cheng, Ming-Guang Feng, and Zhicheng Shen. "Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins." Applied and Environmental Microbiology 73, no. 3 (November 22, 2006): 956–61. http://dx.doi.org/10.1128/aem.02079-06.

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ABSTRACT Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

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Choi, Young J., J. Lawrence Gringorten, Louise Bélanger, Lyne Morel, Denis Bourque, Luke Masson, Denis Groleau, and Carlos B. Míguez. "Production of an Insecticidal Crystal Protein from Bacillus thuringiensis by the Methylotroph Methylobacterium extorquens." Applied and Environmental Microbiology 74, no. 16 (June 13, 2008): 5178–82. http://dx.doi.org/10.1128/aem.00598-08.

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ABSTRACT The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P mxaF . Transmission electron microscopy revealed characteristic bipyramidal intracellular δ-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.

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Chakroun, Maissa, Núria Banyuls, Yolanda Bel, Baltasar Escriche, and Juan Ferré. "Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria." Microbiology and Molecular Biology Reviews 80, no. 2 (March 2, 2016): 329–50. http://dx.doi.org/10.1128/mmbr.00060-15.

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SUMMARYEntomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

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Khandelwal, Puneet, Devapriya Choudhury, Ajanta Birah, M. K. Reddy, Gorakh Prasad Gupta, and Nirupama Banerjee. "Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila." Journal of Bacteriology 186, no. 19 (October 1, 2004): 6465–76. http://dx.doi.org/10.1128/jb.186.19.6465-6476.2004.

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ABSTRACT Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

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Park, Seong-Cheol, Jin-Young Kim, Jong-Kook Lee, Hye Song Lim, Hyosuk Son, Su-Hyang Yoo, Seong-Eun Mun, Mi-Kyeong Jang, and Jung Ro Lee. "Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains." Antibiotics 10, no. 12 (December 20, 2021): 1558. http://dx.doi.org/10.3390/antibiotics10121558.

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Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

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Journal articles on the topic 'Insecticidal protein'

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