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Spodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III β-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.
Isabel Gómez; Josue Ocelotl; Jorge Sánchez; Sotero Aguilar-Medel; Guadalupe Peña-Chora; Laura Lina-Garcia; Alejandra Bravo; Mario Soberón. Bacillus thuringiensis Cry1Ab Domain III β-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith). Applied and Environmental Microbiology 2020, 86, 1 .
AMA StyleIsabel Gómez, Josue Ocelotl, Jorge Sánchez, Sotero Aguilar-Medel, Guadalupe Peña-Chora, Laura Lina-Garcia, Alejandra Bravo, Mario Soberón. Bacillus thuringiensis Cry1Ab Domain III β-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith). Applied and Environmental Microbiology. 2020; 86 (22):1.
Chicago/Turabian StyleIsabel Gómez; Josue Ocelotl; Jorge Sánchez; Sotero Aguilar-Medel; Guadalupe Peña-Chora; Laura Lina-Garcia; Alejandra Bravo; Mario Soberón. 2020. "Bacillus thuringiensis Cry1Ab Domain III β-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith)." Applied and Environmental Microbiology 86, no. 22: 1.
Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II–III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.
Sabino Pacheco; Jean Piere Jesus Quiliche; Isabel Gómez; Jorge Sánchez; Mario Soberón; Alejandra Bravo. Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity. Toxins 2020, 12, 647 .
AMA StyleSabino Pacheco, Jean Piere Jesus Quiliche, Isabel Gómez, Jorge Sánchez, Mario Soberón, Alejandra Bravo. Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity. Toxins. 2020; 12 (10):647.
Chicago/Turabian StyleSabino Pacheco; Jean Piere Jesus Quiliche; Isabel Gómez; Jorge Sánchez; Mario Soberón; Alejandra Bravo. 2020. "Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity." Toxins 12, no. 10: 647.
Scarabaeoidea and Chrysomeloidea insects are agriculture-destructive coleopteran pests. Few effective Bacillus thuringiensis (Bt) insecticidal proteins against these species have been described. Bt isolate BtSU4 was found to be active against coleopteran insects. Genome sequencing revealed two new cry8 genes in BtSU4, designated as cry8Ha1 and cry8Ia1. Both genes expressed a 135 kDa protoxin forming irregular shape crystals. Bioassays performed with Cry8Ha1 protoxin showed that it was toxic to both larvae and adult stages of Holotrichia parallela, also to Holotrichia oblita adults and to Anoplophora glabripennis larvae, but was not toxic to larval stages of H. oblita or Colaphellus bowringi. The Cry8Ia1 protoxin only showed toxicity against H. parallela larvae. After activation with chymotrypsin, the Cry8Ha1 activated toxin lost its insecticidal activity against H. oblita adults and reduced its activity on H. parallela adults, but gained toxicity against C. bowringi larvae, a Chrysomeloidea insect pest that feeds on crucifer crops. The chymotrypsin activated Cry8Ia1 toxin did not show toxicity to any one of these insects. These data show that Cry8Ha1 and Cry8Ia1 protoxin and activated toxin proteins have differential toxicity to diverse coleopteran species, and that protoxin is a more robust protein for the control of coleopteran insects.
Changlong Shu; Guixin Yan; Shizhi Huang; Yongxin Geng; Mario Soberón; Alejandra Bravo; Lili Geng; Jie Zhang. Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Differential Specificity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily. Toxins 2020, 12, 642 .
AMA StyleChanglong Shu, Guixin Yan, Shizhi Huang, Yongxin Geng, Mario Soberón, Alejandra Bravo, Lili Geng, Jie Zhang. Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Differential Specificity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily. Toxins. 2020; 12 (10):642.
Chicago/Turabian StyleChanglong Shu; Guixin Yan; Shizhi Huang; Yongxin Geng; Mario Soberón; Alejandra Bravo; Lili Geng; Jie Zhang. 2020. "Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Differential Specificity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily." Toxins 12, no. 10: 642.
It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.
Jianfeng Zhang; Minghui Jin; Yanchao Yang; Leilei Liu; Yongbo Yang; Isabel Gómez; Alejandra Bravo; Mario Soberón; Yutao Xiao; Kaiyu Liu. The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda. Toxins 2020, 12, 375 .
AMA StyleJianfeng Zhang, Minghui Jin, Yanchao Yang, Leilei Liu, Yongbo Yang, Isabel Gómez, Alejandra Bravo, Mario Soberón, Yutao Xiao, Kaiyu Liu. The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda. Toxins. 2020; 12 (6):375.
Chicago/Turabian StyleJianfeng Zhang; Minghui Jin; Yanchao Yang; Leilei Liu; Yongbo Yang; Isabel Gómez; Alejandra Bravo; Mario Soberón; Yutao Xiao; Kaiyu Liu. 2020. "The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda." Toxins 12, no. 6: 375.
Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.
Lijun Gong; Shi Kang; Junlei Zhou; Dan Sun; Le Guo; Jianying Qin; Liuhong Zhu; Yang Bai; Fan Ye; Mazarin Akami; Qingjun Wu; Shaoli Wang; Baoyun Xu; Zhongxia Yang; Alejandra Bravo; Mario Soberón; Zhaojiang Guo; Lizhang Wen; YouJun Zhang; Guo. Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.). Toxins 2020, 12, 76 .
AMA StyleLijun Gong, Shi Kang, Junlei Zhou, Dan Sun, Le Guo, Jianying Qin, Liuhong Zhu, Yang Bai, Fan Ye, Mazarin Akami, Qingjun Wu, Shaoli Wang, Baoyun Xu, Zhongxia Yang, Alejandra Bravo, Mario Soberón, Zhaojiang Guo, Lizhang Wen, YouJun Zhang, Guo. Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.). Toxins. 2020; 12 (2):76.
Chicago/Turabian StyleLijun Gong; Shi Kang; Junlei Zhou; Dan Sun; Le Guo; Jianying Qin; Liuhong Zhu; Yang Bai; Fan Ye; Mazarin Akami; Qingjun Wu; Shaoli Wang; Baoyun Xu; Zhongxia Yang; Alejandra Bravo; Mario Soberón; Zhaojiang Guo; Lizhang Wen; YouJun Zhang; Guo. 2020. "Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.)." Toxins 12, no. 2: 76.
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.
Paulina Anaya; Janette Onofre; Mary Carmen Torres-Quintero; Jorge Sánchez; Sarjeet S. Gill; Alejandra Bravo; Mario Soberón. Oligomerization is a key step for Bacillus thuringiensis Cyt1Aa insecticidal activity but not for toxicity against red blood cells. Insect Biochemistry and Molecular Biology 2020, 119, 103317 .
AMA StylePaulina Anaya, Janette Onofre, Mary Carmen Torres-Quintero, Jorge Sánchez, Sarjeet S. Gill, Alejandra Bravo, Mario Soberón. Oligomerization is a key step for Bacillus thuringiensis Cyt1Aa insecticidal activity but not for toxicity against red blood cells. Insect Biochemistry and Molecular Biology. 2020; 119 ():103317.
Chicago/Turabian StylePaulina Anaya; Janette Onofre; Mary Carmen Torres-Quintero; Jorge Sánchez; Sarjeet S. Gill; Alejandra Bravo; Mario Soberón. 2020. "Oligomerization is a key step for Bacillus thuringiensis Cyt1Aa insecticidal activity but not for toxicity against red blood cells." Insect Biochemistry and Molecular Biology 119, no. : 103317.
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.
Blanca I. García-Gómez; Sayra N. Cano; Erika E. Zagal; Edgar Dantán-Gonzalez; Alejandra Bravo; Mario Soberón. Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation. mBio 2019, 10, 1 .
AMA StyleBlanca I. García-Gómez, Sayra N. Cano, Erika E. Zagal, Edgar Dantán-Gonzalez, Alejandra Bravo, Mario Soberón. Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation. mBio. 2019; 10 (6):1.
Chicago/Turabian StyleBlanca I. García-Gómez; Sayra N. Cano; Erika E. Zagal; Edgar Dantán-Gonzalez; Alejandra Bravo; Mario Soberón. 2019. "Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation." mBio 10, no. 6: 1.
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.
Wei Wei; Shuang Pan; Yuemin Ma; Yutao Xiao; Yongbo Yang; Sijia He; Alejandra Bravo; Mario Soberón; Kaiyu Liu. GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility. Insect Biochemistry and Molecular Biology 2019, 118, 103306 .
AMA StyleWei Wei, Shuang Pan, Yuemin Ma, Yutao Xiao, Yongbo Yang, Sijia He, Alejandra Bravo, Mario Soberón, Kaiyu Liu. GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility. Insect Biochemistry and Molecular Biology. 2019; 118 ():103306.
Chicago/Turabian StyleWei Wei; Shuang Pan; Yuemin Ma; Yutao Xiao; Yongbo Yang; Sijia He; Alejandra Bravo; Mario Soberón; Kaiyu Liu. 2019. "GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility." Insect Biochemistry and Molecular Biology 118, no. : 103306.
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. armigera 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.
Yuemin Ma; Jianfeng Zhang; Yutao Xiao; Yang; Liu; Rong Peng; Alejandra Bravo; Mario Soberón; Ma; Xiao; Peng; Yanchao Yang; Chenxi Liu; Yongbo Yang; Kaiyu Liu. The Cadherin Cry1Ac Binding-Region is Necessary for the Cooperative Effect with ABCC2 Transporter Enhancing Insecticidal Activity of Bacillus thuringiensis Cry1Ac Toxin. Toxins 2019, 11, 538 .
AMA StyleYuemin Ma, Jianfeng Zhang, Yutao Xiao, Yang, Liu, Rong Peng, Alejandra Bravo, Mario Soberón, Ma, Xiao, Peng, Yanchao Yang, Chenxi Liu, Yongbo Yang, Kaiyu Liu. The Cadherin Cry1Ac Binding-Region is Necessary for the Cooperative Effect with ABCC2 Transporter Enhancing Insecticidal Activity of Bacillus thuringiensis Cry1Ac Toxin. Toxins. 2019; 11 (9):538.
Chicago/Turabian StyleYuemin Ma; Jianfeng Zhang; Yutao Xiao; Yang; Liu; Rong Peng; Alejandra Bravo; Mario Soberón; Ma; Xiao; Peng; Yanchao Yang; Chenxi Liu; Yongbo Yang; Kaiyu Liu. 2019. "The Cadherin Cry1Ac Binding-Region is Necessary for the Cooperative Effect with ABCC2 Transporter Enhancing Insecticidal Activity of Bacillus thuringiensis Cry1Ac Toxin." Toxins 11, no. 9: 538.
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.
Isabel Gómez; Josue Ocelotl; Jorge Sánchez; Christina Lima; Erica Martins; Anayeli Rosales-Juárez; Sotero Aguilar-Medel; André Abad; Hua Dong; Rose Monnerat; Guadalupe Peña; Jie Zhang; Mark Nelson; Gusui Wu; Alejandra Bravo; Mario Soberón. Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability. Applied and Environmental Microbiology 2018, 84, e01393-18 .
AMA StyleIsabel Gómez, Josue Ocelotl, Jorge Sánchez, Christina Lima, Erica Martins, Anayeli Rosales-Juárez, Sotero Aguilar-Medel, André Abad, Hua Dong, Rose Monnerat, Guadalupe Peña, Jie Zhang, Mark Nelson, Gusui Wu, Alejandra Bravo, Mario Soberón. Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability. Applied and Environmental Microbiology. 2018; 84 (20):e01393-18.
Chicago/Turabian StyleIsabel Gómez; Josue Ocelotl; Jorge Sánchez; Christina Lima; Erica Martins; Anayeli Rosales-Juárez; Sotero Aguilar-Medel; André Abad; Hua Dong; Rose Monnerat; Guadalupe Peña; Jie Zhang; Mark Nelson; Gusui Wu; Alejandra Bravo; Mario Soberón. 2018. "Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability." Applied and Environmental Microbiology 84, no. 20: e01393-18.
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 .
Isabel Gómez; Daniel E. Rodríguez-Chamorro; Gabriela Flores-Ramírez; Ricardo Grande; Fernando Zúñiga; Francisco J. Portugal; Jorge Sánchez; Sabino Pacheco; Alejandra Bravo; Mario Soberón. Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin. Applied and Environmental Microbiology 2018, 84, e01089-18 .
AMA StyleIsabel Gómez, Daniel E. Rodríguez-Chamorro, Gabriela Flores-Ramírez, Ricardo Grande, Fernando Zúñiga, Francisco J. Portugal, Jorge Sánchez, Sabino Pacheco, Alejandra Bravo, Mario Soberón. Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin. Applied and Environmental Microbiology. 2018; 84 (17):e01089-18.
Chicago/Turabian StyleIsabel Gómez; Daniel E. Rodríguez-Chamorro; Gabriela Flores-Ramírez; Ricardo Grande; Fernando Zúñiga; Francisco J. Portugal; Jorge Sánchez; Sabino Pacheco; Alejandra Bravo; Mario Soberón. 2018. "Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin." Applied and Environmental Microbiology 84, no. 17: e01089-18.
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, since 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 the domain II-loop3 of Cry9Aa and to 428TKKMKTL434 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.
Zeyu Wang; Longfa Fang; Zishan Zhou; Sabino Pacheco; Isabel Gómez; Fuping Song; Mario Soberón; Jie Zhang; Alejandra Bravo. Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis). Journal of Biological Chemistry 2018, 293, 11447 -11458.
AMA StyleZeyu Wang, Longfa Fang, Zishan Zhou, Sabino Pacheco, Isabel Gómez, Fuping Song, Mario Soberón, Jie Zhang, Alejandra Bravo. Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis). Journal of Biological Chemistry. 2018; 293 (29):11447-11458.
Chicago/Turabian StyleZeyu Wang; Longfa Fang; Zishan Zhou; Sabino Pacheco; Isabel Gómez; Fuping Song; Mario Soberón; Jie Zhang; Alejandra Bravo. 2018. "Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis)." Journal of Biological Chemistry 293, no. 29: 11447-11458.
Pathogenic bacteria use different strategies to infect their hosts including the simultaneous production of pore forming toxins and several virulence factors that help to synergize their pathogenic effects. However, how the pathogenic bacteria are able to complete their life cycle and break out the host intestinal barrier is poorly understood. The infectious cycle ofBacillus thuringiensis(Bt) bacterium inCaenorhabditis elegansis a powerful model system to study the early stages of the infection process. Bt produces Cry pore-forming toxins during the sporulation phase that are key virulence factors involved in Bt pathogenesis. Here we show that during the early stages of infection, the Cry toxins disrupt the midgut epithelial tissue allowing the germination of spores. The vegetative Bt cells then trigger a quorum sensing response that is activated by PlcR regulator resulting in production of different virulence factors, such as the metalloproteinases ColB and Bmp1, that besides Cry toxins are necessary to disrupt the nematode epithelial junctions causing efficient bacterial host infection and dead of the nematode. Overall our work describes a novel mechanism for Bt infection, targeting the epithelial junctions of its host midgut cells.Author summaryThe entomopathogenic bacteriaBacillus thuringiensis(Bt) are used worldwide as biopesticides due to their insecticidal properties. Crystal proteins (Cry) produced by Bt during the sporulation phase of growth are mainly responsible for their insecticidal properties. The infection process of Bt includes three successive steps, virulence, necrotrophic, and sporulation processes. During the virulence process, after ingestion by the susceptible hosts, the Cry toxins form pores in the apical membrane of intestinal cells, inducing favorable conditions for bacterial spore germination. Vegetative bacteria multiply in the host and coordinate their behavior by using the quorum sensor regulator PlcR, which leads to the production of virulence factors allowing the bacteria to kill the host. However, how the bacteria are able to disrupt the host intestinal barrier during the early stages of infection remains unknown. Here we show that Bt employs the nematicidal Cry toxins and additional virulence factors controlled by the PlcR regulon to disrupt the intestinal epithelial junctions ofC. elegansat the early stages of infection allowing that Bt bacteria complete its life cycle in the worms. Our work provides new insights into the pathogenesis of Bt, and highlights the importance of breaking down host epithelial junctions for a successful infection, a similar mechanism could be used by other pathogens-host interactions since epithelial junctions are conserved structures from insects to mammals.
Liting Wan; Jian Lin; Hongwen Du; Alejandra Bravo; Mario Soberón; Donghai Peng; Ming Sun. Bacillus thuringiensistargets the host intestinal epithelial junctions for successful infection ofCaenorhabditis elegans. 2018, 338236 .
AMA StyleLiting Wan, Jian Lin, Hongwen Du, Alejandra Bravo, Mario Soberón, Donghai Peng, Ming Sun. Bacillus thuringiensistargets the host intestinal epithelial junctions for successful infection ofCaenorhabditis elegans. . 2018; ():338236.
Chicago/Turabian StyleLiting Wan; Jian Lin; Hongwen Du; Alejandra Bravo; Mario Soberón; Donghai Peng; Ming Sun. 2018. "Bacillus thuringiensistargets the host intestinal epithelial junctions for successful infection ofCaenorhabditis elegans." , no. : 338236.
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.
Mario Soberón; Leivi Portugal; Blanca-Ines Garcia-Gómez; Jorge Sánchez; Janette Onofre; Isabel Gómez; Sabino Pacheco; Alejandra Bravo. Cell lines as models for the study of Cry toxins from Bacillus thuringiensis. Insect Biochemistry and Molecular Biology 2017, 93, 66 -78.
AMA StyleMario Soberón, Leivi Portugal, Blanca-Ines Garcia-Gómez, Jorge Sánchez, Janette Onofre, Isabel Gómez, Sabino Pacheco, Alejandra Bravo. Cell lines as models for the study of Cry toxins from Bacillus thuringiensis. Insect Biochemistry and Molecular Biology. 2017; 93 ():66-78.
Chicago/Turabian StyleMario Soberón; Leivi Portugal; Blanca-Ines Garcia-Gómez; Jorge Sánchez; Janette Onofre; Isabel Gómez; Sabino Pacheco; Alejandra Bravo. 2017. "Cell lines as models for the study of Cry toxins from Bacillus thuringiensis." Insect Biochemistry and Molecular Biology 93, no. : 66-78.
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.
Wen-Bo Chen; Guo-Qing Lu; Hong-Mei Cheng; Chen-Xi Liu; Yu-Tao Xiao; Chao Xu; Zhi-Cheng Shen; Mario Soberón; Alejandra Bravo; Kong-Ming Wu. Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm. Transgenic Research 2017, 26, 763 -774.
AMA StyleWen-Bo Chen, Guo-Qing Lu, Hong-Mei Cheng, Chen-Xi Liu, Yu-Tao Xiao, Chao Xu, Zhi-Cheng Shen, Mario Soberón, Alejandra Bravo, Kong-Ming Wu. Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm. Transgenic Research. 2017; 26 (6):763-774.
Chicago/Turabian StyleWen-Bo Chen; Guo-Qing Lu; Hong-Mei Cheng; Chen-Xi Liu; Yu-Tao Xiao; Chao Xu; Zhi-Cheng Shen; Mario Soberón; Alejandra Bravo; Kong-Ming Wu. 2017. "Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm." Transgenic Research 26, no. 6: 763-774.
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.
Diana L. Martínez De Castro; Blanca I. García-Gómez; Isabel Gómez; Alejandra Bravo; Mario Soberón. Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda. Peptides 2017, 98, 99 -105.
AMA StyleDiana L. Martínez De Castro, Blanca I. García-Gómez, Isabel Gómez, Alejandra Bravo, Mario Soberón. Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda. Peptides. 2017; 98 ():99-105.
Chicago/Turabian StyleDiana L. Martínez De Castro; Blanca I. García-Gómez; Isabel Gómez; Alejandra Bravo; Mario Soberón. 2017. "Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda." Peptides 98, no. : 99-105.
Bacillus thuringiensis (Bt) are Gram-positive bacteria that produce different insecticidal proteins, named Cry, Vip, and Cyt, during the sporulation phase of growth. Here we will describe each one of these classes of protein, their mechanism of action, and their three-dimensional structure if it is available. We will also describe the different strategies that have been used to find novel insecticidal genes that could be used in biological control of insect pests as well as the strategies to evolve known genes to produce proteins with improved toxicity against selected insect pests. These novel strategies include site-directed mutagenesis and domain swapping among different Cry toxins where novel hybrid proteins containing domains or loop regions from different Cry proteins were constructed, resulting in improved toxicity against selected insect pests. Finally we will describe high-throughput systems that have been used to evolve Cry toxins in vitro. Overall, Bt toxins represent one of the most successful strategies for the biocontrol of insect pests.
Alejandra Bravo; Sabino Pacheco; Isabel Gómez; Blanca Garcia-Gómez; Janette Onofre; Mario Soberón. Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action. Bacillus thuringiensis and Lysinibacillus sphaericus 2017, 53 -66.
AMA StyleAlejandra Bravo, Sabino Pacheco, Isabel Gómez, Blanca Garcia-Gómez, Janette Onofre, Mario Soberón. Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action. Bacillus thuringiensis and Lysinibacillus sphaericus. 2017; ():53-66.
Chicago/Turabian StyleAlejandra Bravo; Sabino Pacheco; Isabel Gómez; Blanca Garcia-Gómez; Janette Onofre; Mario Soberón. 2017. "Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action." Bacillus thuringiensis and Lysinibacillus sphaericus , no. : 53-66.
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.
Josue Ocelotl; Jorge Sánchez; Isabel Gómez; Bruce E. Tabashnik; Alejandra Bravo; Mario Soberón. ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth. Scientific Reports 2017, 7, 2386 .
AMA StyleJosue Ocelotl, Jorge Sánchez, Isabel Gómez, Bruce E. Tabashnik, Alejandra Bravo, Mario Soberón. ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth. Scientific Reports. 2017; 7 (1):2386.
Chicago/Turabian StyleJosue Ocelotl; Jorge Sánchez; Isabel Gómez; Bruce E. Tabashnik; Alejandra Bravo; Mario Soberón. 2017. "ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth." Scientific Reports 7, no. 1: 2386.
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.
Janette Onofre; Meztlli Gaytán; Arlen Peña-Cardeña; Blanca I. García-Gomez; Sabino Pacheco; Isabel Gómez; Alejandra Bravo; Mario Soberón. Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta. Peptides 2017, 98, 93 -98.
AMA StyleJanette Onofre, Meztlli Gaytán, Arlen Peña-Cardeña, Blanca I. García-Gomez, Sabino Pacheco, Isabel Gómez, Alejandra Bravo, Mario Soberón. Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta. Peptides. 2017; 98 ():93-98.
Chicago/Turabian StyleJanette Onofre; Meztlli Gaytán; Arlen Peña-Cardeña; Blanca I. García-Gomez; Sabino Pacheco; Isabel Gómez; Alejandra Bravo; Mario Soberón. 2017. "Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta." Peptides 98, no. : 93-98.
Note: In lieu of an abstract, this is an excerpt from the first page.Excerpt The authors would like to indicate that Dr. Angeles Cancino-Rodezno and Leidy P. Bedoya-Pérez participated equally in their paper published in the International Journal of Molecular Sciences [1].
Leidy P. Bedoya-Pérez; Angeles Cancino-Rodezno; Biviana Flores-Escobar; Mario Soberón; Alejandra Bravo. Addendum: Bedoya-Pérez, L.P. et al. Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis. Int. J. Mol. Sci. 2013, 14, 8467–8478. International Journal of Molecular Sciences 2016, 17, 2021 .
AMA StyleLeidy P. Bedoya-Pérez, Angeles Cancino-Rodezno, Biviana Flores-Escobar, Mario Soberón, Alejandra Bravo. Addendum: Bedoya-Pérez, L.P. et al. Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis. Int. J. Mol. Sci. 2013, 14, 8467–8478. International Journal of Molecular Sciences. 2016; 17 (12):2021.
Chicago/Turabian StyleLeidy P. Bedoya-Pérez; Angeles Cancino-Rodezno; Biviana Flores-Escobar; Mario Soberón; Alejandra Bravo. 2016. "Addendum: Bedoya-Pérez, L.P. et al. Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis. Int. J. Mol. Sci. 2013, 14, 8467–8478." International Journal of Molecular Sciences 17, no. 12: 2021.