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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacillus thuringiensis (Bt) Cyt1Aa toxin shows toxicity to mosquitoes, to certain coleopteran pests and also to red blood cells (RBC). However, its mode of action in the different target cells is not well defined. This protein is a single α-β domain pore-forming toxin, where a β sheet is wrapped by two α-helices layers. The Cyt1Aa α-helix hairpin in the N-terminal has been proposed to be involved in initial membrane binding and oligomerization, while the β sheet inserts into the membrane to form a pore that lyze the cells. To determine the role of the N-terminal α-helix hairpin region of Cyt1Aa in its mode of action, we characterized different single point mutations located in helices α-1 and α-2. Eight cysteine substitutions in different residues were produced in Bt, and we found that three of them: Cyt1AaA65C, Cyt1AaL85C and Cyt1AaN89C, lost insecticidal toxicity against Aedes aegypti larvae but retained similar or increased hemolytic activity towards rabbit RBC. Analysis of toxin binding and oligomerization using Ae. aegypti midgut brush border membrane vesicles showed that the three Cyt1Aa mutants non-toxic to Ae. aegypti were affected in oligomerization. However, these mutants were still hemolytic. Our data shows that oligomerization of Cyt1Aa toxin is essential for its toxicity to Ae. aegypti but not for its toxicity against RBC indicating that the mode of action of Cyt1Aa is different in these distinct target membranes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells. IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance., (Copyright © 2020 American Society for Microbiology.)

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells., Competing Interests: Declaration of competing interest M. S. and A. B. are coauthors of a patent on modified Bt toxins. “Suppression of resistance in insects to Bacillus thuringiensis Cry toxins, using toxins that do not required the cadherin receptor” (patent numbers: CA2690188A1, CN101730712A, EP2184293A2, EP2184293A4, EP2184293B1, WO2008150150A2, WO2008150150A3)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCE Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins., (Copyright © 2019 García-Gómez et al.)

Bacillus thuringiensis Cry1Ac toxin binds to midgut proteins, as cadherin (CAD) and ABCC2 transporter, to form pores leading to larval death. In cell lines, co-expression of CAD and ABCC2 enhance Cry1Ac toxicity significantly, but the mechanism remains elusive. Here, we show that the expression of Helicoverpa armigera CAD (HaCAD-GFP) in Hi5 cells induces susceptibility to Cry1Ac and enhanced Cry1Ac toxicity when co-expressed with H. armig era ABCC2 (HaABCC2-GFP), since Cry1Ac toxicity increased 735-fold compared to Hi5 cells expressing HaCAD-GFP alone or 28-fold compared to HaABCC2-GFP alone. In contrast, the expression of the Spodoptera litura CAD (SlCAD-GFP) in Hi5 cells did not induce susceptibility to Cry1Ac nor it potentiated Cry1Ac toxicity with HaABCC2-GFP. To identify the CAD regions involved in the enhancement of Cry1Ac toxicity with ABCC2, the different CAD domains were replaced between SlCAD-GFP and HaCad-GFP proteins, and cytotoxicity assays were performed in Hi5 cells in the absence or presence of HaABCC2-GFP. The HaCAD toxin-binding region (TB), specifically the CAD repeat-11, was necessary to enhance Cry1Ac toxicity with ABCC2. We propose that CAD TB is involved in recruiting Cry1Ac to localize it in a good position for its interaction with the ABCC2, resulting in efficient toxin membrane insertion enhancing Cry1Ac toxicity.

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., (© 2018 Peña-Cardeña et al.)

The western corn rootworm (WCR) Diabrotica virgifera virgifera causes substantial damage in corn. Genetically modified (GM) plants expressing some Bacillus thuringiensis (Bt) insecticidal Cry proteins efficiently controlled this pest. However, changes in WCR susceptibility to these Bt traits have evolved and identification of insecticidal proteins with different modes of action against WCR is necessary. We show here for the first time that Cyt1Aa from Bt exhibits toxicity against WCR besides to the dipteran Aedes aegypti larvae. Cyt1Aa is a pore-forming toxin that shows no cross-resistance with mosquitocidal Cry toxins. We characterized different mutations in helix α-A from Cyt1Aa. Two mutants (A61C and A59C) exhibited reduced or absent hemolytic activity but retained toxicity to A. aegypti larvae, suggesting that insecticidal and hemolytic activities of Cyt1Aa are independent activities. These mutants were still able to form oligomers in synthetic lipid vesicles and to synergize Cry11Aa toxicity. Remarkably, mutant A61C showed a five-fold increase insecticidal activity against mosquito and almost 11-fold higher activity against WCR. Cyt1Aa A61C mutant was as potent in killing WCR that were selected for resistance to mCry3A as it was against unselected WCR indicating that this toxin could be a useful resistance management option in the control of WCR.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars., Competing Interests: The authors have declared that no competing interests exist.

Bacillus thuringiensis Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. Spodoptera frugiperda is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for S. frugiperda control. The β16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab β16 ( 509 STLRVN 514 ) revealed that certain β16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for S. frugiperda without affecting the toxicity for other lepidopteran larvae, such as Manduca sexta larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity toward S. frugiperda A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four S. frugiperda populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of five S. frugiperda populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the S. frugiperda populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for S. frugiperda ALP, APN, and cadherin receptors. IMPORTANCE Spodoptera frugiperda is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Bacillus thuringiensis Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of S. frugiperda , although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for S. frugiperda without loss of toxicity to Manduca sexta Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of S. frugiperda and other lepidopteran pests in the field., (Copyright © 2018 American Society for Microbiology.)

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 Q 125 from SfABCC2 or E 125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors. Consistently with this, cells expressing Helicoverpa armigera HaABCC2 Q122 -GFP, were more susceptible to Cry1Ac than cells expressing HaABCC2 E122 -GFP mutant. Q 125 or E 125 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacillus thuringiensis Cry1Ca is toxic to different Spodoptera species. The aims of this work were to identify the Cry1Ca-binding proteins in S. frugiperda , to provide evidence on their participation in toxicity, and to identify the Cry1Ca amino acid residues involved in receptor binding. Pulldown assays using Spodoptera frugiperda brush border membrane vesicles (BBMV) identified aminopeptidase N (APN), APN1, and APN2 isoforms as Cry1Ca-binding proteins. Cry1Ca alanine substitutions in all residues of domain III β16 were characterized. Two β16 nontoxic mutants (V505A and S506A) showed a correlative defect on binding to the recombinant S. frugiperda APN1 (SfAPN1). Finally, silencing the expression of APN1 transcript, by double-stranded RNA (dsRNA) feeding, showed that silenced larvae are more tolerant of the Cry1Ca toxin, which induced less than 40% mortality in silenced larvae whereas nonsilenced larvae had 100% mortality. Overall, our results show that Cry1Ca relies on APN1 binding through domain III β16 to impart toxicity to S. frugiperda IMPORTANCE Bacillus thuringiensis Cry toxins rely on receptor binding to exert toxicity. Cry1Ca is toxic to different populations of S. frugiperda , a major corn pest in America. Nevertheless, the S. frugiperda midgut proteins that are involved in Cry1Ca toxicity have not been identified. Here we identified aminopeptidase N1 (APN1) as a functional receptor of Cry1Ca. Moreover, we showed that Cry1Ca domain III β16 is involved in APN1 binding. These results give insights on potential target sites for improving Cry1Ca toxicity to S. frugiperda ., (Copyright © 2018 American Society for Microbiology.)

The bacterium Bacillus thuringiensis produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two B. thuringiensis toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer ( Chilo suppressalis ). We found that these two proteins bind specifically to brush border membrane vesicles of C. suppressalis and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and to 428 TKKMKTL 434 in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the B. thuringiensis Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among B. thuringiensis toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance., (© 2018 Wang et al.)

Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.

Cell lines have been use extensively for the study of the mode of action of different pore forming toxins produced by different bacterial species. Bacillus thuringiensis Cry toxins are not the exception and their mechanism of action has been analyzed in different cell lines. Here we review the data obtained with different cell lines, including those that are naturally susceptible to the three domain Cry toxins (3d-Cry) and other non-susceptible cell lines that have been transformed with 3d-Cry toxin binding molecules cloned from the susceptible insects. The effects on Cry toxin action after expressing different insect gut proteins, such as glycosyl-phosphatidyl-inositol (GPI) anchored proteins (like alkaline phosphatase (ALP) aminopeptidase (APN)), or trans-membrane proteins (like cadherin (CAD) or ATP-binding cassette subfamily C member 2 (ABCC2) transporter) in cell lines showed that, with few exceptions, expression of GPI-anchored proteins do not correlated with increased susceptibility to the toxin, while the expression of CAD or ABCC2 proteins correlated with induced susceptibility to Cry toxins in the transformed cells lines. Also, that the co-expression of CAD and ABCC2 transporter induced a synergistic effect in the toxicity of 3d-Cry toxins. Overall the data show that in susceptible cell lines, the 3d-Cry toxins induce pore formation that correlates with toxicity. However, the intracellular responses remain controversial since it was shown that the same 3d-Cry toxin in different cell lines activated different responses such as adenylate cyclase-PKA death response or apoptosis. Parasporins are Cry toxins that are toxic to cancer cell lines that have structural similarities with the insecticidal Cry toxins. They belong to the 3d-Cry toxin or to MTX-like Cry toxin families but also show important differences with the insecticidal Cry proteins. Some parasporins are pore-forming toxins, and some activate apoptosis. In this review we summarized the results of the different studies about the Cry toxins mode of action using cultured cell lines and discuss their relation with the studies performed in insect larvae., (Copyright © 2017. Published by Elsevier Ltd.)

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the β-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73 - resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis , Plutella xylostella , or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants. IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers ( Laodelphax striatellus ) and white-backed planthoppers ( Sogatella furcifera ). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management., (Copyright © 2018 American Society for Microbiology.)

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity., (Copyright © 2017 American Society for Microbiology.)

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes ( cry8 -like and cry8Ga ), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita , and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum , ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of H. oblita and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against H. oblita larvae., (Copyright © 2017 American Society for Microbiology.)

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm ( Helicoverpa armigera ), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A 548 to S 798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera IMPORTANCE: Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity., (Copyright © 2017 American Society for Microbiology.)

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K + ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K + ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive insect pest of corn in China. Susceptibility to the Cry1F toxin derived from Bacillus thuringiensis has been demonstrated for ACB, suggesting the potential for Cry1F inclusion as part of an insect pest management program. Insects can develop resistance to Cry toxins, which threatens the development and use of Bt formulations and Bt crops in the field. To determine possible resistance mechanisms to Cry1F, a Cry1F-resistant colony of ACB (ACB-FR) that exhibited more than 1700-fold resistance was established through selection experiments after 49 generations of selection under laboratory conditions. The ACB-FR strain showed moderate cross-resistance to Cry1Ab and Cry1Ac of 22.8- and 26.9-fold, respectively, marginally cross-resistance to Cry1Ah (3.7-fold), and no cross-resistance to Cry1Ie (0.6-fold). The bioassay responses of progeny from reciprocal F1 crosses to different Cry1 toxin concentrations indicated that the resistance trait to Cry1Ab, Cry1Ac and Cry1F has autosomal inheritance with no maternal effect or sex linked. The effective dominance (h) of F1 offspring was calculated at different concentrations of Cry1F, showing that h decreased as concentration of Cry1F increased. Finally, the analysis of actual and expected mortality of the progeny from a backcross (F1 × resistant strain) indicated that the inheritance of the resistance to Cry1F in ACB-FR was due to more than one locus. The present study provides an understanding of the genetic basis of Cry1F resistance in ACB-FR and also shows that pyramiding Cry1F with Cry1Ah or Cry1Ie could be used as a strategy to delay the development of ACB resistance to Bt proteins.

Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Asian corn borer (ACB), Ostrinia furnacalis, is an important insect pest of maize susceptible to different Cry1A toxins. Based on amino acid sequence alignment of ALP sequences from lepidopteran larvae an alp gene was cloned from ACB, named ofalp. Pull dawn assays using biotinylated Cry1Ac and brush border membrane vesicles isolated from second instar ACB larvae showed that four proteins of 50, 65, 68 and 70kDa precipitated with the Cry1Ac. The 65kDa band cross-reacted with the anti-OfALP monoclonal antibody. GalNac was able to release the binding of Cry1Ac to the 65kDa OfALP in pull down assays. A 37kDa fragment from residues D173 to D473 of OfALP was cloned and expressed in Escherichia coli cells. We show that this ALP-fragment was able to bind Cry1Ac in ligand blot analysis. Our data also indicate that different ALP isoforms or variants may be also Cry1Ac binding proteins since more ALP enzymatic activity was pull down with Cry1Ac than with anti-OfALP antibody. We also analyzed the expression levels of ALP throughout the larval development by qPCR and ALP enzymatic activity. Our data indicated that ALP expression in ACB was observed preferentially in young instar larvae. Finally, we show that resistance in O. furnacalis ACB-AcR strain resistant to Cry1Ac did not correlate with changes in expression of this ALP protein since it shows similar gene expression of ofalp than the susceptible insect strain. Identification of Cry1Ac receptors will help to understand mechanism of action of Cry1Ac in O. furnacalis and to understand mechanism of Cry toxin resistance. Our data indicate that at least one ALP protein is involved in the binding interaction with Cry1Ac in O. furnacalis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.

Background: Although much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion., Results: In this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses., Conclusions: This study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.

Insecticidal proteins from Bacillus thuringiensis (Bt) are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV) from resistant and susceptible larvae of Pectinophora gossypiella (pink bollworm), a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers in vitro without cadherin and killed P. gossypiella larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant P. gossypiella larvae that have cadherin mutations.

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.

Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To exert their toxic effect, Bacillus thuringiensis Cry1Ab toxin undergoes a sequential binding mechanism with different larval gut proteins including glycosyl-phosphatidyl-inositol anchored proteins like aminopeptidase-N (APN) or alkaline-phosphatase (ALP) and a transmembrane cadherin to form pre-pore structures that insert into the membrane. Cadherin binding induces oligomerization of the toxin by facilitating removal of the N-terminal region, while APN/ALP binding helps in oligomer membrane insertion. Cry1AbMod toxin was engineered to lack N-terminal region of the toxin and shown to counter resistance linked to cadherin mutations. In this manuscript we determined the toxicity of Cry1AbMod to Manduca sexta larvae silenced in the expression of cadherin, ALP or APN receptors. As previously reported Cry1Ab toxicity relied principally in ALP and cadherin in comparison to APN. Our data shows that Cry1AbMod counters resistance associated with low cadherin expression but was not effective against ALP silenced larvae. These results show that Cry1AbMod could be effective against resistance insects linked to mutations on binding molecules involved in toxin oligomerization but not against resistant insects linked to mutations on binding molecules involved in oligomer membrane insertion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.

Bacillus thuringiensis Cry toxins are insecticidal proteins used to control insect pests. The interaction of Cry toxins with the midgut of susceptible insects is a dynamic process involving activation of the toxin, binding to midgut receptors in the apical epithelium and conformational changes in the toxin molecule, leading to pore formation and cell lysis. An understanding of the molecular events underlying toxin mode of action is essential for the continued use of Cry toxins. In this work, we examined the mechanism of action of Cry1A toxins in the lepidopteran cell line CF-1, using native Cry1Ab and mutant forms of this protein that interfer with different steps in the mechanism of action, specifically, receptor binding, oligomerization or pore formation. These mutants lost activity against both Manduca sexta larvae and CF-1 cells. We also analyzed a mutation created in domain I of Cry1Ab, in which helix α-1 and part of helix α-2 were deleted (Cry1AbMod). Cry1AbMod is able to oligomerize in the absence of toxin receptors, and although it shows reduced activity against some susceptible insects, it kills insect pests that have developed resistance to native Cry1Ab. Cry1AbMod showed enhanced toxicity to CF-1, suggesting that oligomerization of native Cry1Ab may be a limiting step in its activity against CF-1 cells. The toxicity of Cry1Ac and Cry1AcMod were also analyzed. Our results suggest that some of the steps in the mode of action of Cry1A toxins are conserved in vivo in insect midgut cells and in vitro in an established cell line, CF-1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.

Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.

Bacillus thuringiensis produces insecticidal Cry and Cyt proteins that are toxic to different insect orders. In addition, Cyt toxins also display haemolytic activity. Both toxins are pore-forming proteins that form oligomeric structures that insert into the target membrane to lyse cells. Cyt toxins play an important role in mosquitocidal activity since they synergize Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes, and this interaction is important to induce the synergistic activity observed. It was proposed that Cyt toxins do not interact with protein receptors but directly interacting with the specific midgut cell lipids. Here, we analysed if oligomerization and membrane insertion of Cyt1Aa are necessary steps to synergize Cry11Aa toxicity. We characterized Cyt1Aa helix α-C mutants that were affected in oligomerization, in membrane insertion and also in haemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11Aa toxicity indicating these steps are independent events of Cyt1Aa synergistic activity. Furthermore, the data indicate that formation of stable Cyt1Aa-oligomeric structure is a key step for membrane insertion, haemolysis and insecticidal activity., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. Cells can trigger different survival mechanisms to counteract the effects of sub-lytic doses of pore forming toxins. Particularly, two signaling pathways have been demonstrated to play a role in the defense mechanism to other toxins in Caenorhabditis elegans and in mammalian cells. These are the unfolded protein response (UPR) and the sterol regulatory element binding proteins (SREBP) pathways, which are proposed to facilitate membrane repair responses. In this work we analyzed the role of these pathways in Aedes aegypti response to intoxication with Cry11Aa toxin. We show that UPR is activated upon toxin ingestion. The role of these two pathways was analyzed in vivo by using RNA interference. We silenced the expression of specific proteins in A. aegypti larvae. Gene silencing of Ire-1 and Xbp-1 proteins from UPR system, resulted in hypersensitive to Cry11Aa toxin action. In contrast, silencing of Cas-1, Scap and S2P from SREBP pathway had no affect on Cry11Aa toxicity in A. aegypti larvae. However, the role of SREBP pathway requires further studies to be conclusive. Our data indicate that the UPR pathway is involved in the insect defense against Cry toxins.

Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures., (© 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)