scholarly journals Spodoptera frugiperda (J. E. Smith) Aminopeptidase N1 Is a Functional Receptor of the Bacillus thuringiensis Cry1Ca Toxin

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Isabel Gómez ◽  
Daniel E. Rodríguez-Chamorro ◽  
Gabriela Flores-Ramírez ◽  
Ricardo Grande ◽  
Fernando Zúñiga ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Receptor Binding ◽  
Spodoptera Frugiperda ◽  
Binding Proteins ◽  
Membrane Vesicles ◽  
Amino Acid Residues ◽  
Content Type ◽  
Different Populations ◽  
Domain Iii ◽  
Functional Receptor

ABSTRACT 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.

scholarly journals Enhancement ofBacillus thuringiensisCry1Ab and Cry1Fa Toxicity toSpodoptera frugiperdaby Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Isabel Gómez ◽  
Josue Ocelotl ◽  
Jorge Sánchez ◽  
Christina Lima ◽  
Erica Martins ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Spodoptera Frugiperda ◽  
Transgenic Maize ◽  
Invasive Pest ◽  
Effective Control ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Lepidopteran Pests ◽  
Domain Iii

ABSTRACTBacillus thuringiensisCry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests.Spodoptera frugiperdais 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 forS. frugiperdacontrol. 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 (509STLRVN514) revealed that certain β16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab forS. frugiperdawithout affecting the toxicity for other lepidopteran larvae, such asManduca sextalarvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity towardS. frugiperda. A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against fourS. frugiperdapopulations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of fiveS. frugiperdapopulations. In contrast, Cry1AbN514F showed increased toxicity in only two of theS. frugiperdapopulations 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 forS. frugiperdaALP, APN, and cadherin receptors.IMPORTANCESpodoptera frugiperdais the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled byBacillus thuringiensisCry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control ofS. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity forS. frugiperdawithout loss of toxicity toManduca 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 ofS. frugiperdaand other lepidopteran pests in the field.

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

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Isabel Gómez ◽  
Josue Ocelotl ◽  
Jorge Sánchez ◽  
Sotero Aguilar-Medel ◽  
Guadalupe Peña-Chora ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Insect Pest ◽  
Transgenic Maize ◽  
Crop Damage ◽  
Binding Interaction ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Lepidopteran Pests ◽  
Domain Iii

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

scholarly journals Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Jian Jiang ◽  
Ying Huang ◽  
Changlong Shu ◽  
Mario Soberón ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Binding Proteins ◽  
Insect Pest ◽  
Membrane Vesicles ◽  
Economic Losses ◽  
Binding Assays ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Lepidopteran Insect

ABSTRACT 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.

scholarly journals Role of Bacillus thuringiensis Toxin Domains in Toxicity and Receptor Binding in the Diamondback Moth

1999 ◽  
Vol 65 (5) ◽  
pp. 1900-1903 ◽  
Author(s):  
V. Ballester ◽  
F. Granero ◽  
R. A. de Maagd ◽  
D. Bosch ◽  
J. L. Ménsua ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Receptor Binding ◽  
Diamondback Moth ◽  
Membrane Vesicles ◽  
Binding Assays ◽  
Structural Domains ◽  
Specificity Of Binding ◽  
Domain Iii ◽  
Domain I

ABSTRACT The toxic fragment of Bacillus thuringiensis crystal proteins consists of three distinct structural domains. There is evidence that domain I is involved in pore formation and that domain II is involved in receptor binding and specificity. It has been found that, in some cases, domain III is also important in determining specificity. Furthermore, involvement of domain III in binding has also been reported recently. To investigate the role of toxin domains in the diamondback moth (Plutella xylostella), we used hybrid toxins with domain III substitutions among Cry1C, Cry1E, and Cry1Ab. Neither Cry1E nor G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) was toxic, whereas Cry1C and F26 (the reciprocal hybrid) were equally toxic. H04 (a hybrid with domains I and II from Cry1Ab and domain III from Cry1C) showed toxicity that was of a similar level as that of Cry1Ab and significantly higher than that of Cry1C. Binding assays with 125I-Cry1C showed that Cry1C and F26 competed for the same binding sites on midgut membrane vesicles, whereas Cry1E, G27, and H04 did not bind to these sites. Our results show that, in contrast to findings in other insects for the toxins and hybrids used here, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I) and not by domain III.

scholarly journals Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

2004 ◽  
Vol 384 (3) ◽  
pp. 507-513 ◽  
Author(s):  
Salvador HERRERO ◽  
Joel GONZÁLEZ-CABRERA ◽  
Juan FERRÉ ◽  
Petra L. BAKKER ◽  
Ruud A. de MAAGD
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Exigua ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Oligomeric Form ◽  
Specific Receptors ◽  
Domain Iii

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541–544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

scholarly journals Specific Binding of Radiolabeled Cry1Fa Insecticidal Protein from Bacillus thuringiensis to Midgut Sites in Lepidopteran Species

2012 ◽  
Vol 78 (11) ◽  
pp. 4048-4050 ◽  
Author(s):  
Carmen Sara Hernández-Rodríguez ◽  
Patricia Hernández-Martínez ◽  
Jeroen Van Rie ◽  
Baltasar Escriche ◽  
Juan Ferré
Keyword(s):  
Spodoptera Frugiperda ◽  
Heliothis Virescens ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Insecticidal Protein ◽  
Content Type ◽  
Lepidopteran Species ◽  
Equilibrium Binding ◽  
Competition Assays

ABSTRACTCry1Fa insecticidal protein was successfully radiolabeled with125I-Na. Specific binding to brush border membrane vesicles was shown for the lepidopteran speciesOstrinia nubilalis,Spodoptera frugiperda,Spodoptera exigua,Helicoverpa armigera,Heliothis virescens, andPlutella xylostella. Homologous competition assays were performed to obtain equilibrium binding parameters (Kd[dissociation constant] andRt[concentration of binding sites]) for these six insect species.

Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda

Peptides ◽  
2017 ◽  
Vol 98 ◽  
pp. 99-105 ◽  
Author(s):  
Diana L. Martínez de Castro ◽  
Blanca I. García-Gómez ◽  
Isabel Gómez ◽  
Alejandra Bravo ◽  
Mario Soberón
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Binding Proteins

scholarly journals Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

2008 ◽  
Vol 74 (9) ◽  
pp. 2565-2572 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Insect Larvae ◽  
Insecticidal Toxin ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

scholarly journals Bacillus thuringiensisCry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms,Spodoptera frugiperda(J.E. Smith)

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Yanfei Wang ◽  
Jinling Wang ◽  
Xiaoran Fu ◽  
Jeffrey R. Nageotte ◽  
Jennifer Silverman ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Protein Interactions ◽  
Spodoptera Frugiperda ◽  
Insect Cells ◽  
Fall Armyworm ◽  
Insecticidal Protein ◽  
Row Crops ◽  
Content Type ◽  
Resistance Risk ◽  
Important Pest

ABSTRACTTwo new modifiedBacillus thuringiensis(Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW),Spodoptera frugiperda(J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially availableBtproteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCEThere is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW),Spodoptera frugiperda, a maize pest that already has developed resistance toBacillus thuringiensis(Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially availableBtproteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

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