Biosynthesis of Pteridines in Insects: A Review.

Pteridines are important cofactors for many biological functions of all living organisms, and they were first discovered as pigments of insects, mainly in butterfly wings and the eye and body colors of insects. Most of the information on their structures and biosynthesis has been obtained from studies with the model insects Drosophila melanogaster and the silkworm Bombyx mori. This review discusses, and integrates into one metabolic pathway, the different branches which lead to the synthesis of the red pigments "drosopterins", the yellow pigments sepiapterin and sepialumazine, the orange pigment erythropterin and its related yellow metabolites (xanthopterin and 7-methyl-xanthopterin), the colorless compounds with violet fluorescence (isoxanthopterin and isoxantholumazine), and the branch leading to tetrahydrobiopterin, the essential cofactor for the synthesis of aromatic amino acids and biogenic amines.

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.

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis.

Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin-activated form involves a major conformational change of the N-terminal Domain I, which is remodelled into a tetrameric coiled-coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C-t Domains IV and V to sustain the conformational change necessary for toxicity.

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis.

Due to their different specificity, the use of Vip3 proteins from Bacillus thuringiensis in combination with the conventionally used Cry proteins in crop protection is being essential to counteract the appearance of insect resistance. Therefore, understanding the mode of action of Vip3 proteins is crucial for their better application, with special interest on the binding to membrane receptors as the main step for specificity. Derived from in vitro heterologous competition binding assays using 125I-Vip3A and other Vip3 proteins as competitors, it has been shown that Vip3 proteins share receptors in Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). In this study, using 125I-Vip3Aa, we have first extended the in vitro competition binding site model of Vip3 proteins to Spodoptera littoralis. With the aim to understand the relevance (in terms of toxicity) of the binding to the midgut sites observed in vitro on the insecticidal activity of these proteins, we have performed in vivo competition assays with S. littoralis larvae, using disabled mutant (non-toxic) Vip3 proteins as competitors for blocking the toxicity of Vip3Aa and Vip3Af. The results of the in vivo competition assays confirm the occurrence of shared binding sites among Vip3 proteins and help understand the functional role of the shared binding sites as revealed in vitro.

Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis.

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Comparison of in vitro and in vivo binding site competition of Bacillus thuringiensis Cry1 proteins in two important maize pests.

BACKGROUND: Binding site models, derived from in vitro competition binding studies, have been widely used for predicting potential cross-resistance among insecticidal proteins from Bacillus thuringiensis. However, because discrepancies have been found between binding data and observed cross-resistance patterns in some insect species, new tools are required to study the functional relevance of the shared binding sites. RESULTS: Here, an in vivo approach has been applied to the competition studies to establish the functional relevance of shared binding sites as determined by in vitro competition assays. Using Cry disabled proteins as competitors in mixed protein overlay assays, we assessed the preference of Cry1Ab, Cry1Fa, and Cry1A.105 proteins for shared binding sites in vivo in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda. CONCLUSION: This study shows that in vivo and in vitro binding site competition assays can provide useful information to better ascertain whether different Cry proteins share binding sites and, consequently, whether cross-resistance due to binding site alteration can occur. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultured Insect Cells.

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125I-DI-III. In addition, the truncated protein 125I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo.IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors.

Laboratory selection for resistance of field populations is a well-known and useful tool to understand the potential of insect populations to evolve resistance to insecticides. It provides us with estimates of the frequency of resistance alleles and allows us to study the mechanisms by which insects developed resistance to shed light on the mode of action and optimize resistance management strategies. Here, a field population of Mythimna separata was subjected to laboratory selection with either Vip3Aa, Cry1Ab, or Cry1F insecticidal proteins from Bacillus thuringiensis. The population rapidly evolved resistance to Vip3Aa reaching, after eight generations, a level of >3061-fold resistance, compared with the unselected insects. In contrast, the same population did not respond to selection with Cry1Ab or Cry1F. The Vip3Aa resistant population did not show cross resistance to either Cry1Ab or Cry1F. Radiolabeled Vip3Aa was tested for binding to brush border membrane vesicles from larvae from the susceptible and resistant insects. The results did not show any qualitative or quantitative difference between both insect samples. Our data, along with previous results obtained with other Vip3Aa-resistant populations from other insect species, suggest that altered binding to midgut membrane receptors is not the main mechanism of resistance to Vip3Aa.

Hetero-oligomerization of Bacillus thuringiensis Cry1A proteins enhance binding to the ABCC2 transporter of Spodoptera exigua.

The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labeling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.

Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used in sprayable formulations or produced in transgenic crops as the most successful alternatives to synthetic pesticides. The most relevant threat to sustainability of Bt insecticidal proteins (toxins) is the evolution of resistance in target pests. To date, high-level resistance to Bt sprays has been limited to one species in the field and another in commercial greenhouses. In contrast, there are currently seven lepidopteran and one coleopteran species that have evolved practical resistance to transgenic plants producing insecticidal Bt proteins. In this article, we present a review of the current knowledge on mechanisms of resistance to Bt toxins, with emphasis on key resistance genes and field-evolved resistance, to support improvement of Bt technology and its sustainability.

Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins.

Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives is bioinsecticides based on Bacillus thuringiensis This entomopathogenic bacterium produces different protein types which are toxic to several insect, mite, and nematode species. Currently, insecticidal proteins belonging to the Cry and Vip3 groups are widely used to control insect pests both in formulated sprays and in transgenic crops. However, the benefits of B. thuringiensis-based products are threatened by insect resistance evolution. Numerous studies have highlighted that mutations in genes coding for surrogate receptors are responsible for conferring resistance to B. thuringiensis Nevertheless, other mechanisms may also contribute to the reduction of the effectiveness of B. thuringiensis-based products for managing insect pests and even to the acquisition of resistance. Here, we review the relevant literature reporting how invertebrates (mainly insects and Caenorhabditis elegans) respond to exposure to B. thuringiensis as either whole bacteria, spores, and/or its pesticidal proteins.

Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis.

Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 protein by site-directed mutagenesis, distributed along the five structural domains of the protein. Ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). The results showed that, to render a wild type fragment pattern upon trypsin treatment, position 483 required an acidic residue, and position 552 an aromatic residue. Regarding toxicity, the change of Met34 to Lys34 significantly increased the toxicity of the protein for one of the three insect species tested (S. littoralis), whereas the other residue substitutions did not improve, or even decreased, insect toxicity, confirming their key role in the structure/function of the protein.

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action.

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date [...].

Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis.

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.

Evaluation of the Toxicity of Supernatant Cultures and Spore-Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70 kDa. The LC-MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce ß-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach.

Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides.IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests.

The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodopteraexigua, Spodopteralittoralis, Spodopterafrugiperda,Helicoverpaarmigera, Mamestrabrassicae, Anticarsiagemmatalis, and Ostriniafurnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S.frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189-509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostriniafurnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.

Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants.

Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of the trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning of Vip3Af, we selected mutants with an altered proteolytic pattern. Based on protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains which match some of the domains proposed independently by two in silico models. Domain I ranges amino acids (aa) 12-198, domain II aa199-313, domain III aa314-526, domain IV aa527-668, and domain V aa669-788. The effect of some mutations on the ability to form a tetrameric molecule revealed that domains I-II are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and destroy the tetrameric form upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to the Vip3 family of proteins.