Bacillus Thuringiensis Cry Toxins Research Articles

Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 μg/mL - 60.70 μg/mL, and an IC50 of 10.65 μg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Group Selection as a Basis for Screening Mutagenized Libraries of Public Goods (Bacillus thuringiensis Cry Toxins).

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Baseline susceptibility of cabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) to Bacillus thuringiensis (Berliner) Cry toxins in Meghalaya

BackgroundCabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae), is one of the most important pests of cabbage and other cruciferous crops and accounts for > 40% yield reduction in the crops. An investigation on the baseline susceptibility of P. brassicae to Bacillus thuringiensis (Bt) Cry toxins in Meghalaya was evaluated for future exploitation in Bt resistance monitoring. Two different Cry toxins, Bt Cry1C and Cry2Ab, were screened against 11 different field populations of P. brassicae from the state of Meghalaya. LC50 was evaluated based on the response of larval mortality of P. brassicae using the leaf-dip bioassay method.ResultsThe baseline-susceptibility tests conducted on P. brassicae in 11 different field populations from Meghalaya revealed that Smit population strains seemed to show less tolerance to both the Bt Cry toxins (Cry1C and Cry2Ab). Compared to the Cry1C toxin, Cry2Ab was found more potent against P. brassicae. The median lethal concentrations, LC50 72 h, varied from 0.535 to 1.725 µg/ml for Cry2Ab and 0.546–1.803 µg/ml for Cry1C toxin. The screening using leaf-dip bioassay resulted in a tolerance ratio of 3.3-fold and 3.2-fold for Cry1C and Cry2Ab, respectively. The most tolerant strains of P. brassicae from Umiam and Pepbah regions were observed to show discriminating concentrations of 19.30 µg/ml for Cry1C and 24.03 µg/ml for Cry2Ab (LC99, 72 h).ConclusionsThe Cry2Ab toxin was found to be more virulent than Cry1C toxin for P. brassicae. Certain candidate discriminating concentrations for Cry1C and Cry2Ab can be used as benchmarks for future resistance monitoring of P. brassicae to Bt Cry toxins.

Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody.

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16μg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21μM and 1.24μM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

Effectiveness of the natural resistance management refuge for Bt-cotton is dominated by local abundance of soybean and maize

Genetically engineered crops expressing Bacillus thuringiensis (Bt) Cry toxins have transformed insect management in maize and cotton, reducing insecticide use and associated off-target effects. To mitigate the risk that pests evolve resistance to Bt crops, the US Environmental Protection Agency requires resistance management measures. The approved resistance management plan for Bt maize in cotton production regions requires a structured refuge of non-Bt maize equal to 20% of the maize planted; that for Bt cotton relies on the presence of an unstructured natural refuge comprising both non-Bt crop and non-crop hosts. We examined how abundance of Bt crops (cotton and maize) and an important non-Bt crop (soybean) component of the natural refuge affect resistance to Bt Cry1Ac toxin in local populations of Helicoverpa zea, an important lepidopteran pest impacted by Bt cotton and maize. We show refuge effectiveness is responsive to local abundances of maize and cotton and non-Bt soybean, and maize, in its role as a source of H. zea infesting cotton and non-Bt hosts, influences refuge effectiveness. These findings have important implications for commercial and regulatory decisions regarding deployment of Bt toxins targeting H. zea in maize, cotton, and other crops and for assumptions regarding efficacy of natural refuges.

Pore-forming protein complexes from Pleurotus mushrooms kill western corn rootworm and Colorado potato beetle through targeting membrane ceramide phosphoethanolamine

Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.

The C-terminal protoxin region of Bacillus thuringiensis Cry1Ab toxin has a functional role in binding to GPI-anchored receptors in the insect midgut

Bacillus thuringiensis Cry toxins are used worldwide for controlling insects. Cry1Ab is produced as a 130-kDa protoxin that is activated by proteolytic removal of an inert 500 amino-acid-long C-terminal region, enabling the activated toxin to bind to insect midgut receptor proteins, leading to its membrane insertion and pore formation. It has been proposed that the C-terminal region is only involved in toxin crystallization, but its role in receptor binding is undefined. Here we show that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) insect receptors. ELISA, ligand blot, surface plasmon resonance, and pulldown assays revealed that the Cry1Ab C-terminal region binds to both ALP and APN but not to cadherin. Thus, the C-terminal region provided a higher binding affinity of the protoxin to the gut membrane that correlated with higher toxicity of protoxin than activated toxin. Moreover, Cry1Ab domain II loop 2 or 3 mutations reduced binding of the protoxin to cadherin but not to ALP or APN, supporting the idea that protoxins have additional binding sites. These results imply that two different regions mediate the binding of Cry1Ab protoxin to membrane receptors, one located in domain II-III of the toxin and another in its C-terminal region, suggesting an active role of the C-terminal protoxin fragment in the mode of action of Cry toxins. These results suggest that future manipulations of the C-terminal protoxin region could alter the specificity and increase the toxicity of B. thuringiensis proteins.

A single amino acid polymorphism in ABCC2 loop 1 is responsible for differential toxicity of Bacillus thuringiensis Cry1Ac toxin in different Spodoptera (Noctuidae) species

Bacillus thuringiensis Cry toxins exert their toxicity by forming membrane pores after binding with larval midgut membrane proteins known as receptors. Spodoptera litura and Spodoptera frugiperda belong to the same genus, but S. litura is tolerant to Cry1Ac, while S. frugiperda is susceptible. The mechanism involved in the differential toxicity of Cry1Ac to these insect species is not understood. Amino acid sequences analysis of ABCC2, a well-recognized Cry1Ac receptor, from both species showed high sequence identity. Hi5 insect cells expressing SfABCC2 from S. frugiperda were 65-fold more susceptible than those expressing the SlABCC2 from S. litura. Substitution of fragments, point mutations and deletions between the ABCC2 of the two species revealed that ABCC2 amino acid Q125 from SfABCC2 or E125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors. Consistently with this, cells expressing Helicoverpa armigera HaABCC2Q122-GFP, were more susceptible to Cry1Ac than cells expressing HaABCC2E122-GFP mutant. Q125 or E125 is located in a predicted exposed loop 1 region of ABCC2 indicating that this region could be important for Cry1Ac binding. These findings identified a single amino acid residue located in loop 1 of ABCC2 transporter as responsible for the different levels of susceptibility to Cry1Ac among various lepidopteran species.

Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins

Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.

Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis

Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7–CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7–CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7–CR11 was constructed by homology modeling. Molecular docking results of CR7–CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7–CR11, while CR7–CR8 was the region of CR7–CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7–CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Summary Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med‐fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well‐known as invertebrate‐active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N‐terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid‐cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda

Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The LC–MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda.

Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mutagenesis and screening

The cultivation of genetically modified crops (GMCs) has greatly increased worldwide. Given the fact that over 10 Bacillus thuringiensis Cry toxins have been applied in GMCs, there is a need to develop an efficient and economically affordable detection method that simultaneously screen such compounds with similar structures in crops and foodstuff. Here we described an approach using a site-directed mutagenesis that enhances the generic specificity of single chain variable fragment (scFv). After three rounds of panning against mixed antigen (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F) from the constructed mutagenesis library, one mutant showed greater ability to bind to a range of Cry toxins. The phage mutant, named D9, was cloned into pET26b expression vector, and then induced and purified. The mutant D9 was used to develop an indirect competitive immunoassay that sucessfully recognizes five targeted Cry1 toxins with a working range from 0.20 to 2.22μgmL−1. The recoveries of five Cry toxins from spiked rice samples ranged from 86.67% to 96.67%, with a coefficient of variation less than 8%. The results suggest that the immunoassay based on a scFv is a promising approach for detection of Cry toxins with a broad specificity.

Knockdown of two Cadherin genes confers resistance to Cry2A and Cry1C in Chilo suppressalis

Bacillus thuringiensis (Bt) Cry toxins play an important role in the management of insect pests. Resistance to Bt toxins has been reported in many pest insects but the mechanism responsible for this resistance in rice crop pests remains largely unknown. Cadherin is one of several Bt toxin receptors. At present, only one cadherin gene, CsCAD1, has been documented in the striped rice stem borer, Chilo suppressalis. We amplified a nearly full-length transcript of another C. suppressalis cadherin gene, CsCAD2, and found that it has a different expression pattern to CsCAD1. CsCAD1 was highly expressed in fifth and sixth instar larvae, especially in the midgut, while the expression levels of CsCA2 were equably in each developmental stage. Newly hatched larvae were fed on rice smeared with synthesized siRNA to knockdown either CsCAD1 or CsCAD2, and then were fed transgenic rice expressing either the Cry2A or Cry1C toxins. The siRNA-treatment groups had lower mortality and higher survival rates than the control group, suggesting that reduced expression of CsCAD1 or CsCAD2 increased resistance to Cry2A and Cry1C. We conclude that CsCAD1 and CsCAD2 interact with Bt toxins in C. suppressalis and that this interaction could be the mechanism underlying Bt resistance in this insect.

Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters

In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.

RNA interference knockdown of aminopeptidase N genes decrease the susceptibility of Chilo suppressalis larvae to Cry1Ab/Cry1Ac and Cry1Ca-expressing transgenic rice

Transgenic rice expressing Bacillus thuringiensis (Bt) Cry toxins are resistant to lepidopteran pests, such as Chilo suppressalis, a major insect pest of rice in Asia. Understanding how these toxins interact with their hosts is crucial to understanding their insecticidal action. In this study, knockdown of two aminopeptidase N genes (APN1 and APN2) by RNA interference resulted in decreased susceptibility of C. suppressalis larvae to the Bt rice varieties TT51 (Cry1Ab and Cry1Ac fusion genes) and T1C-19 (Cry1Ca), but not T2A-1 (Cry2Aa). This suggests that APN1 and APN2 are receptors for Cry1A and Cry1C toxins in C. suppressalis.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A-C16A mutant did not formed oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A-C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Loop replacements with gut-binding peptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens (Stål).

Bacillus thuringiensis (Bt) Cry toxins have been used widely in pest managements. However, Cry toxins are not effective against sap-sucking insects (Hemiptera), which limits the application of Bt for pest management. In order to extend the insecticidal spectrum of Bt toxins to the rice brown planthopper (BPH), Nilaparvata lugens, we modified Cry1Ab putative receptor binding domains with selected BPH gut-binding peptides (GBPs). Three surface exposed loops in the domain II of Cry1Ab were replaced with two GBPs (P2S and P1Z) respectively. Bioassay results showed that toxicity of modified toxin L2-P2S increased significantly (~9 folds) against BPH nymphs. In addition, damage of midgut cells was observed from the nymphs fed with L2-P2S. Our results indicate that modifying Cry toxins based on the toxin-gut interactions can broaden the insecticidal spectrum of Bt toxin. This method provides another approach for the development of transgenic crops with novel insecticidal activity against hemipteran insects and insect populations resistant to current Bt transgenic crops.