Cry Proteins Research Articles

Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Objective: Pain conditions exhibit variations linked to circadian rhythms. Circadian rhythms, regulated by clock proteins, impact melatonin levels and immune functions. Melatonin, structurally similar to indomethacin, a non-steroidal anti-inflammatory drug, serves as an alternative in pain-related conditions. Literature suggests a correlation between melatonin-dependent regulation of circadian rhythm and reductions in pain complaints. 2(3<i>H</i>)-Benzoxazolone, known for its anti-inflammatory and analgesic properties, is a promising scaffold for drug design. In this study, pharmacophore analysis focused on benzoxazolone derivatives, evaluating their impact on clock proteins, and providing insights into potential chronotherapeutic implications. Methods: Molecular docking and dynamics simulations were conducted on CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 clock proteins using benzoxazolone and its 5-substituted derivatives. Molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) calculations were employed to analyze binding free energies. Benzoxazolone derivatives, especially 5-nitro-2-benzoxazolone and 5-fluoro-2-benzoxazolone, exhibited elevated binding affinities on clock proteins compared to melatonin and indomethacin, the reference molecules. The interaction profiles and stability of complexes were maintained throughout the simulations. Results: The results suggest a regulatory impact on CRY proteins, emphasizing their role in circadian rhythm and pain modulation. In addition, the benzoxazolone ring and its derivatives, structurally resembling the core structure of melatonin and indomethacin, demonstrate promising affinities on clock proteins. Conclusion: These findings provide preliminary data and hypothetically propose benzoxazolone derivatives as potential candidates for dual functionality in analgesic activity and circadian rhythm regulation, warranting further in vitro and clinical investigations.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12–9.86 μg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66–20.46 μg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36–36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Cry Toxins from <i>Bt</i> Bollgard II® Cotton Seeds: An Affordable Source for Insect Bioassays

The relative expression of Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab toxins from seeds of Bt Bollgard II® cotton was quantified using Enzyme Linked Immunosorbent Assays (ELISA) method. The ELISA technique involves binding a sample or calibrator to a solid-phase antibody, forming a "sandwich complex" with an enzyme-labeled antibody. The resulting enzyme substrate reaction produces a product proportional to the antigen concentration. The study reveals that each gram of Bt Bollgard II® seed powder contains 3.25 μg Cry1Ac and 182.32 μg Cry2Ab, with Cry2Ab expression 56 times higher. Variability in Cry protein expression is influenced by factors like temperature, genetic background, and plant maturity. The research emphasizes the importance of Bt plants, particularly Bt Bollgard II® seed powder as a valuable source for studying insect responses to combined Cry1Ac + Cry2Ab expression in bioassay studies.

Biocidal Activity of the Bacillus thuringiensis 3D Cry Toxins, Molecular Crosstalk at the Insect Midgut with Implication in Insect Resistance Development

Biocidal Activity of the Bacillus thuringiensis 3D Cry Toxins, Molecular Crosstalk at the Insect Midgut with Implication in Insect Resistance Development

Blue light perceiving CRY proteins: protecting plants from DNA damage.

Blue light perceiving CRY proteins: protecting plants from DNA damage.

Enzyme-linked Immunoassay for the Detection and Quantification of Cry Protein in Bacillus thuringiensis Strains

Enzyme-linked Immunoassay for the Detection and Quantification of Cry Protein in Bacillus thuringiensis Strains

Understanding the Red Shift in the Absorption Spectrum of the FAD Cofactor in ClCry4 Protein.

It is still a puzzle that has not been entirely solved how migratory birds utilize the Earth's magnetic field for biannual migration. The most consistent explanation thus far is rooted in the modulation of the biological function of the cryptochrome 4 (Cry4) protein by an external magnetic field. This phenomenon is closely linked with the flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the protein. Cry4 is activated by blue light, which is absorbed by the FAD cofactor. Subsequent electron and proton transfers trigger radical pair formation in the protein, which is sensitive to the external magnetic field. An important long-lasting redox state of the FAD cofactor is the signaling (FADH•) state, which is present after the transient electron transfer steps have been completed. Recent experimental efforts succeeded in crystallizing the Cry4 protein from Columbia livia (ClCry4) with all of the important residues needed for protein photoreduction. This specific crystallization of Cry4 protein so far is the only avian cryptochrome crystal structure available, which, however, has great similarity to the Cry4 proteins of night migratory birds. The previous experimental studies of the ClCry4 protein included the absorption properties of the protein in its different redox states. The absorption spectrum of the FADH• state demonstrated a peculiar red shift compared to the photoabsorption properties of the FAD cofactor in its FADH• state in other Cry proteins from other species. The aim of this study is to understand this red shift by employing the tools of computational microscopy and, in particular, a QM/MM approach that relies on the polarizable embedding approximation.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa’s different domains could potentially be helpful in the rational design of improved toxins.In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105 + Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic cropsproducing insecticidal proteins fromBacillus thuringiensis Berliner (Bt,Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.)rapidly evolved in some FAW populations, pyramided Bt maizehybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.

The role of GPI-anchored membrane-bound alkaline phosphatase in the mode of action of Bt Cry1A toxins in the diamondback moth

The insecticidal Cry proteins produced by the bacterium Bacillus thuringiensis (Bt) are extensively used for pest control in formulated sprays and in genetically modified crops, but resistance to Bt toxins threatens their sustainable use in agriculture. Understanding the molecular mechanisms involved in Bt pathogenesis is crucial for the development of effective resistance management strategies. Previously, we showed a strong correlation between Cry1Ac resistance in Plutella xylostella (L.) and down-regulation of the glycosylphosphatidylinositol (GPI)-anchored membrane-bound alkaline phosphatase (mALP) and aminopeptidase (APN) and members of the ATP-binding cassette (ABC) transporter subfamily C (ABCC), but we do not yet have a clear understanding of the relative contribution of each midgut receptor type. Here, a P. xylostella strain homozygous for the PxmALP gene knockout was generated using CRISPR/Cas9 and the results showed that this strain had a 294-fold resistance to Cry1Ac toxin and 394-fold cross-resistance to Cry1Ab. Moreover, a triple knockout strain lacking PxmALP, PxABCC2, and PxABCC3 exhibited 9,660-fold resistance to Cry1Ac and 5,662-fold cross-resistance to Cry1Ab. These resistance levels surpassed those observed in the previously described double PxABCC2 and PxABCC3 knockout mutant, revealing a functional redundancy between ABC transporters and PxmALP. In addition, the activity of Cry1A toxins against Sf9 cells expressing PxmALP, PxABCC2 or PxABCC3 confirmed that each of these can act as a functional receptor. Our findings are crucial for unraveling the relative role of multiple receptors and the molecular mechanisms underlying Bt resistance in insects.

Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.

Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Abstract 7221: A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model

Abstract Anti VEGF A antibody Bevacizumab combined with standard chemotherapy has improved outcomes for CRC patients. However, a subset of CRC patients acquires resistance to anti-VEGF treatment and underlying mechanism is poorly understood. Our recent study reported that circadian clock transcription factor BMAL1 drives transcription of VEGFA conferring resistance to bevacizumab treatment. Our rational is to stabilize negative regulator of circadian clock CRY protein to inhibit BMAL1 activity in a negative feedback loop. In this study, we evaluated whether CRY stabilizers SHP656 and SHP1705 in combination with bevacizumab can synergistically enhance anti-tumor effect in CRC mouse models. Methods: In vitro tube formation assay was performed with HUVECs cells to investigate the effect of CRY stabilizer on tube formation. For in vivo experiments, 106 HCT116 human CRC cells were injected subcutaneously into the right flank of 8-week-old nude mice. Mice (5mice/group) were randomized into: A. vehicle (2% carboxymethylcellulose PO 5 days/week; control IgG IP, 2 days/week); B. bevacizumab (5mg/kg IP, twice/week); C. 10mg/kg SHP656; D. 30mg/kg SHP656; E. 30mg/kg SHP1705; F. 10mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP656 + bevacizumab; G. 30mg/kg SHP1705 + bevacizumab. SHP 656 & 1705 was given PO; 5days/week. Tumor volume was measured three times a week. The survival curve was plotted using Kaplan and Meier method and assessed for statistical significance with log-rank test. Results: The in vitro tube formation assay showed significant inhibition of tube formation in HUVEC cells treated with SHP1705 plus bevacizumab compared to treated with bevacizumab alone. In in vivo study, the tumors treated with 30mg/kg SHP1705 + bevacizumab significantly suppressed tumor growth compared to bevacizumab monotherapy (one way-ANOVA, P <0.05). 30mg/kg SHP1705 + bevacizumab significantly improved survival of the mice (Log-rank test, P <0.05). No significant survival benefit was observed with bevacizumab alone. 3/5 mice treated with SHP1705 + bevacizumab showed decreased tumor volume and achieved complete regression for more than 4 months without additional treatment. In combination with bevacizumab, higher dose of SHP656 (30mg/kg) delayed tumor growth and extended survival compared with bevacizumab monotherapy (though did not reach statistical significance) whereas the lower dose of 10mg/kg did not show anti-tumor effect, demonstrating a dose dependent response. Conclusion: Our study shows that CRY stabilizers in combination with bevacizumab significantly enhances anti-tumor efficacy in CRC mouse models. Although mechanism of action of SHP compounds on angiogenesis pathway still needs further investigation, these findings may support development of novel treatment strategies modulating circadian clock to overcome bevacizumab resistance in CRC patients. Citation Format: Jae Ho Lo, Shivani Soni, Goar Smbatyan, Lesly Torres-Gonzalez, Pooja Mittal, Yan Yang, Francesca Battaglin, Priscilla Chan, Yuanzhong Pan, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Indrakant K. Singh, Evanthia T. Roussos Torres, Shannon M. Mumenthaler, Steve A. Kay, Heinz-Josef Lenz. A compound targeting the circadian clock protein CRY2 enhances therapeutic efficacy of bevacizumab in a colorectal cancer (CRC) xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7221.

Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

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

Commentary: Analyzing invertebrate bitopic cadherin G protein-coupled receptors that bind Cry toxins of Bacillus thuringiensis

Commentary: Analyzing invertebrate bitopic cadherin G protein-coupled receptors that bind Cry toxins of Bacillus thuringiensis