Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

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.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Use of RNAi as a preliminary tool for screening putative receptors of nematicidal toxins from Bacillus thuringiensis.

Bacillus thuringiensis is a potential control agent for plant-parasitic nematodes. Nematode intestinal receptors for Cry21-type toxins are poorly known. Therefore, a strategy was tested as a primary screening tool to find possible Cry toxin receptors, using a nematicidal Bt strain and the RNAi technique on Caenorhabditis elegans. Six genes encoding intestinal membrane proteins were selected (abt-4, bre-1, bre-2, bre-3, asps-1, abl-1) as possible targets for Cry proteins. Fractions of each selected gene were amplified by PCR. Amplicons were cloned into the L4440 vector to transform the E. coli HT155 (DE3) strain. Transformed bacteria were used to silence the selected genes using the RNAi feeding method. Nematodes with silenced genes were tested with the Bt strain LBIT-107, which harbors the nematicidal protein Cry21Aa3, among others. Results indicated that nematodes with the silenced abt-4 gene were 69.5% more resistant to the LBIT-107 strain, in general, and 79% to the Cry21Aa3 toxin, specifically.

Criteria to evaluate the reliability of interaction studies with insecticidal proteins.

This paper recommends five criteria to evaluate the reliability of interaction studies with insecticidal proteins. However, these criteria are broadly applicable to an interaction analysis with any type of substance. The recommended criteria reflect the consensus of the literature on interaction analysis from decades of research in fields such as pharmacology and toxicology. The criteria can be used to interrogate the experimental design, assay methodology, data analysis, and interpretation of the results. These criteria will be useful to researchers to help identify the strengths and potential weaknesses of interaction studies and to help define the limits of interpretation of the data. The criteria will also be useful to risk assessors evaluating the reliability of interaction data as part of an environmental risk assessment, and to inform a weight of evidence analysis when there are contradictory results. In addition, these criteria can be used prospectively by researchers to help avoid common pitfalls that are apparent in some interaction studies. Five examples have been provided, with studies from the literature, that demonstrate how these criteria can be objectively and consistently applied to score the reliability of interaction studies with insecticidal proteins that differ in design and methodology.

In Vivo Enzyme Entrapment in a Protein Crystal.

Cry3Aa is a protein that forms crystals naturally in the bacterium Bacillus thuringiensis. Here we report that coexpression of Cry3Aa and a Proteus mirabilis lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst. These characteristics, along with its greatly simplified method of isolation, highlight the potential of Cry3Aa crystal-mediated enzyme entrapment for use in biocatalysis and other biotechnological applications.

Bacillus thuringiensis Cry1Ab Domain III ß-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith).

The fall armyworm, Spodoptera frugiperda, is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III ß-sheets ß-16 and ß-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III ß-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor.IMPORTANCESpodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III ß-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.

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.

Replacement of loop2 and 3 of Cry1Ai in domain II affects specificity to the economically important insect Bombyx mori.

Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity.

Identification of Aedes aegypti specificity motifs in the N-terminus of the Bacillus thuringiensis Cry2Aa pesticidal protein.

One advantage of using the Cry proteins of Bacillus thuringiensis as pesticides is their relatively narrow spectrum of activity, thus reducing the risk of non-target effects. Understanding the molecular basis of specificity has the potential to help us design improved products against emerging pests, or against pests that have developed resistance to other Cry proteins. Many previous studies have associated specificity with the binding of the Cry protein, particularly through the apical regions of domain II, to particular receptors on the midgut epithelial cells of the host insect. We have previously found that the specificity of Cry2A proteins against some insects is associated with domain I, which is traditionally associated with pore-formation but not receptor binding. In this work we identify four amino acids in the N-terminal region that, when mutated, can confer activity towards Aedes aegypti to Cry2Ab, a protein known to lack this toxicity. Intriguingly these amino acids are located in the region (amino acids 1-49) that is believed to be removed during proteolytic activation of the Cry protein. We discuss how the motifs containing these amino acids might be involved in the toxic process.

Immobilization of Bacillus thuringiensis Cry1Ac in metal-organic frameworks through biomimetic mineralization for sustainable pest management.

Traditional chemical pesticide dosage forms and crude application methods have resulted in low pesticide utilization, increased environmental pollution, and the development of resistance. Compared to traditional pesticides, nanopesticides enhance the efficiency of pesticide utilization and reduce the quantity required, thereby decreasing environmental pollution. Herein, Cry1Ac insecticidal crystal protein from Bacillus thuringiensis Subsp. Kurstaki HD-73 was encapsulated in a metal-organic framework (zeolite imidazolate framework-8, ZIF-8) through biomimetic mineralization to obtain Cry1Ac@ZIF-8 nanopesticides. The Cry1Ac@ZIF-8 nanopesticides exhibited a dodecahedral porous structure, and the introduction of Cry1Ac did not affect the intrinsic crystal structure of ZIF-8. The indoor toxicity analysis revealed that the toxicity of Cry1Ac towards Ostrinia furnacalis (Guenée), Helicoverpa armigera Hubner, and Spodoptera litura Fabricius was not affected by ZIF-8 encapsulation. Surprisingly, Cry1Ac@ZIF-8 still exhibited excellent pest management efficacy even after exposure to heat, UV irradiation, and long-term storage. More importantly, the encapsulation of ZIF-8 significantly enhanced the internal absorption performance of Cry1Ac in maize leaves and extended its persistence period. Thus, ZIF-8 could potentially serve as a promising carrier for the preparation of nanopesticides with enhanced applicability, stability, and persistence period, providing a powerful strategy to improve the application of Cry1Ac in future agricultural pest management.

Targeting Design of Human Anti-idiotypic Genetically Engineered Antibody for Simulating the Structure and Insecticidal Function of Bt Cry1C Toxin.

The ß-type anti-Id (Ab2ß) is considered to have potential for simulating the structure and function of the antigen. In this study, a ß-type anti-Id (3A7 anti-I-GEAb) of the Cry1C toxin was captured from a GEAb library. Subsequently, a higher activity of mutant (3A7 mutant 8) was obtained from the mutagenesis library based on 3A7 anti-I-GEAb. The LD50 values of 3A7 anti-I-GEAb and 3A7 mutant 8 reach up to 38.9% and 46.8% of Cry1C toxin for P. xylostella and reach up to 32.9% and 37.4% of Cry1C toxin for H. armigera. Additionally, an IC-ELISA was established based on 3A7 mutant 8 (as the coated "antigen"), with an LOD value of 0.35 ng/mL, exhibiting good accuracy and stability for detecting Cry1C toxin in spiked samples. The present ß-type anti-I-GEAb not only exhibits insecticidal activity similar to Cry1C toxin, offering potential for environmentally friendly pest management, but it can also replace the Cry1C toxin structure to establish a highly sensitive and specific IC-ELISA for monitoring Cry1C toxin.

Death-Associated LIM-Only Protein Reduces Cry1Ac Toxicity by Sequestration of Cry1Ac Protoxin and Activated Toxin in Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

Synergism of Cry1 Toxins by a Fusion Protein Derived from a Cadherin Fragment and an Antibody Peptide.

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

The base and root of domain II loops of Cry toxins contribute to binding to Bombyx mori ABC transporter C2.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.

Genome-wide analysis of V-ATPase genes in Plutella xylostella (L.) and the potential role of PxVHA-G1 in resistance to Bacillus thuringiensis Cry1Ac toxin.

The rapid development of insecticide resistance has hampered the use of Bacillus thuringiensis (Bt), a widely used bio-pesticide. Plutella xylostella (L.) is a globally distributed lepidopteran pest of cruciferous vegetables and has developed severe field resistance to the Bt toxin. Vacuolar H+-ATPases (VHA) are multi-subunit complexes and participate in multiple physiological processes. However, the characterization and functional studies of VHA genes are lacking in insects. This study performed a genome-wide analysis and identified 35 VHA gene family members divided into 15 subfamilies in P. xylostella. We cloned a V-ATPase subunit G gene, PxVHA-G1, in our previous midgut transcriptome profiles. Quantitative reverse transcriptase-polymerase chain reaction results showed that PxVHA-G1 was upregulated in the Cry1S1000-resistant strain than in the G88-susceptible strain, and its expression profile revealed that the midgut, Malpighian tubules, and larva stages generally showed high expression levels. RNAi-mediated knockdown of the PxVHA-G1 gene increased the susceptibility of P. xylostella (G88 and Cry1S1000) to Cry1Ac toxin. Our study is the first to explore the role of PxVHA-G1 on regulating Cry1Ac toxicity in P. xylostella, thus, providing new insights into the role of VHAs in the development of Cry1Ac resistance and sustainable development of pest management.

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

The development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different scientific fields, including toxinology and structural biology, as well as from industry, notably for biotechnological and medical applications. As a natural crystal-making bacterium, Bacillus thuringiensis (Bt) has evolved through millions of years to produce hundreds of highly structurally diverse pesticidal proteins as micrometer-sized crystals. The long-term stability of Bt protein crystals in aqueous environments and their specific and controlled dissolution are characteristics that are particularly sought after. In this article, I explore whether the crystallization machinery of Bt can be hijacked as a means to produce (micro)crystalline formulations of proteins for three different applications: (i) to develop new bioinsecticidal formulations based on rationally improved crystalline toxins, (ii) to functionalize crystals with specific characteristics for biotechnological and medical applications, and (iii) to produce microcrystals of custom proteins for structural biology. By developing the needs of these different fields to figure out if and how Bt could meet each specific requirement, I discuss the already published and/or patented attempts and provide guidelines for future investigations in some underexplored yet promising domains.

How Does Bacillus thuringiensis Crystallize Such a Large Diversity of Toxins?

Bacillus thuringiensis (Bt) is a natural crystal-making bacterium. Bt diversified into many subspecies that have evolved to produce crystals of hundreds of pesticidal proteins with radically different structures. Their crystalline form ensures stability and controlled release of these major virulence factors. They are responsible for the toxicity and host specificity of Bt, explaining its worldwide use as a biological insecticide. Most research has been devoted to understanding the mechanisms of toxicity of these toxins while the features driving their crystallization have long remained elusive, essentially due to technical limitations. The evolution of methods in structural biology, pushing back the limits of the resolution attainable, now allows access to be gained to structural information hidden within natural crystals of such toxins. In this review, I present the main parameters that have been identified as key drivers of toxin crystallization in Bt, notably in the light of recent discoveries driven by structural biology studies. Then, I develop how the future evolution of structural biology will hopefully unveil new mechanisms of Bt toxin crystallization, opening the door to their hijacking with the aim of developing a versatile in vivo crystallization platform of high academic and industrial interest.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP's) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane.

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.