Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

ABSTRACTBacillus thuringiensisCry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genusCylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites inCylas puncticollisto orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu47for the 70-kDa form or Ile88for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) fromC. puncticollislarvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.

Download Full-text

Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

Applied and Environmental Microbiology ◽

10.1128/aem.02791-10 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3182-3188 ◽

Cited By ~ 67

Author(s):

C. Gouffon ◽

A. Van Vliet ◽

J. Van Rie ◽

S. Jansens ◽

J. L. Jurat-Fuentes

Keyword(s):

Bacillus Thuringiensis ◽

Brush Border ◽

Binding Sites ◽

Brush Border Membrane ◽

Specific Binding ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Target Range ◽

Content Type ◽

Bt Toxins

ABSTRACTThe use of combinations ofBacillus thuringiensis(Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species,Heliothis virescens,Helicoverpa zea, andHelicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

Download Full-text

A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

Applied and Environmental Microbiology ◽

10.1128/aem.66.4.1553-1558.2000 ◽

2000 ◽

Vol 66 (4) ◽

pp. 1553-1558 ◽

Cited By ~ 28

Author(s):

Carolina Rausell ◽

Amparo Consuelo Martínez-Ramírez ◽

Inmaculada García-Robles ◽

María Dolores Real

Keyword(s):

Bacillus Thuringiensis ◽

Larval Development ◽

Binding Sites ◽

Specific Binding ◽

Membrane Vesicles ◽

Thaumetopoea Pityocampa ◽

Binding Assays ◽

Forest Pests ◽

First Instar ◽

Nun Moth

ABSTRACT The insecticidal activity and receptor binding properties ofBacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) andLymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.

Download Full-text

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens

Applied and Environmental Microbiology ◽

10.1128/aem.00326-17 ◽

2017 ◽

Vol 83 (11) ◽

Cited By ~ 11

Author(s):

Yolanda Bel ◽

Joel J. Sheets ◽

Sek Yee Tan ◽

Kenneth E. Narva ◽

Baltasar Escriche

Keyword(s):

Bacillus Thuringiensis ◽

Binding Sites ◽

Specific Binding ◽

Management Strategies ◽

Anticarsia Gemmatalis ◽

Cross Resistance ◽

Binding Studies ◽

Content Type ◽

Bt Toxins ◽

Competition Binding

ABSTRACT Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens. Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops. IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Download Full-text

Cry64Ba and Cry64Ca, Two ETX/MTX2-TypeBacillus thuringiensisInsecticidal Proteins Active against Hemipteran Pests

Applied and Environmental Microbiology ◽

10.1128/aem.01996-17 ◽

2017 ◽

Vol 84 (3) ◽

Cited By ~ 10

Author(s):

Yonglei Liu ◽

Yinglong Wang ◽

Changlong Shu ◽

Kejian Lin ◽

Fuping Song ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Specific Binding ◽

Genetically Modified Crops ◽

Membrane Vesicles ◽

Laodelphax Striatellus ◽

Sogatella Furcifera ◽

Cry Toxins ◽

Content Type ◽

Bt Toxin ◽

Rice Planthoppers

ABSTRACTGenetically modified crops that express insecticidalBacillus 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 striatellusandSogatella furcifera. Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the β-pore-forming toxins. Coexpression ofcry64Baandcry64Cagenes in the acrystalliferous Bt strain HD73−resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such asOstrinia furnacalis,Plutella xylostella, orColaphellus 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 fromL. 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.IMPORTANCEIn Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effectiveBacillus 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 thecry64Baandcry64Cagenes 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.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.00541-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 4

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.

Download Full-text

Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus

Journal of Bacteriology ◽

10.1128/jb.00252-17 ◽

2017 ◽

Vol 199 (20) ◽

Cited By ~ 6

Author(s):

Xiating Gao ◽

Yang Liu ◽

Huan Liu ◽

Zhen Yang ◽

Qin Liu ◽

...

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Vibrio Alginolyticus ◽

Physiological Adaptation ◽

Pcr Analysis ◽

Growth Stages ◽

Mobility Shift ◽

Marine Animals ◽

Content Type ◽

Virulence Gene Expression

ABSTRACT In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR. DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus. IMPORTANCE In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in V. alginolyticus, a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the aphB gene itself was identified to be positively autoregulated, and AphB also positively controlled asp and luxR expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.07591-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 4048-4050 ◽

Cited By ~ 9

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.

Download Full-text

Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites

Applied and Environmental Microbiology ◽

10.1128/aem.02342-08 ◽

2009 ◽

Vol 75 (7) ◽

pp. 2236-2237 ◽

Cited By ~ 102

Author(s):

Janete A. D. Sena ◽

Carmen Sara Hernández-Rodríguez ◽

Juan Ferré

Keyword(s):

Bacillus Thuringiensis ◽

Spodoptera Frugiperda ◽

Binding Sites ◽

Specific Binding ◽

Binding Assays ◽

Binding Interactions ◽

Specific Binding Sites

ABSTRACT Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

Download Full-text

Unshared binding sites for Bacillus thuringiensis Cry3Aa and Cry3Ca proteins in the weevil Cylas puncticollis (Brentidae)

Toxicon ◽

10.1016/j.toxicon.2016.09.014 ◽

2016 ◽

Vol 122 ◽

pp. 50-53 ◽

Cited By ~ 1

Author(s):

Patricia Hernández-Martínez ◽

Natalia Mara Vera-Velasco ◽

Baltasar Escriche

Keyword(s):

Bacillus Thuringiensis ◽

Binding Sites ◽

Cylas Puncticollis

Download Full-text

Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.437-442.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 437-442 ◽

Cited By ~ 18

Author(s):

María A. Ibargutxi ◽

Anna Estela ◽

Juan Ferré ◽

Primitivo Caballero

Keyword(s):

Bacillus Thuringiensis ◽

Binding Sites ◽

Specific Binding ◽

Significant Inhibition ◽

The Other ◽

Cross Resistance ◽

Earias Insulana ◽

Cry Proteins ◽

Cry Toxins ◽

Laboratory Colony

ABSTRACT Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. 125I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.

Download Full-text