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

2017 ◽  
Vol 84 (3) ◽  
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.

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

2011 ◽  
Vol 77 (10) ◽  
pp. 3182-3188 ◽  
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.

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

2014 ◽  
Vol 80 (24) ◽  
pp. 7545-7550 ◽  
Author(s):  
Patricia Hernández-Martínez ◽  
Natalia Mara Vera-Velasco ◽  
María Martínez-Solís ◽  
Marc Ghislain ◽  
Juan Ferré ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Sweet Potato ◽  
Binding Sites ◽  
Specific Binding ◽  
Pest Resistance ◽  
Membrane Vesicles ◽  
Sub Saharan Africa ◽  
Receptor Binding Site ◽  
Content Type ◽  
Cylas Puncticollis

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.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT 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.

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.

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

2006 ◽  
Vol 72 (1) ◽  
pp. 437-442 ◽  
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.

scholarly journals Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

2014 ◽  
Vol 81 (5) ◽  
pp. 1884-1890 ◽  
Author(s):  
Wendy Kain ◽  
Xiaozhao Song ◽  
Alida F. Janmaat ◽  
Jian-Zhou Zhao ◽  
Judith Myers ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Trichoplusia Ni ◽  
Resistance Mechanisms ◽  
Vegetable Production ◽  
Bt Cotton ◽  
Content Type ◽  
Bt Toxin ◽  
Bt Toxins ◽  
Cotton Plants ◽  
Incomplete Resistance

ABSTRACTTwo populations ofTrichoplusia nithat had developed resistance toBacillus thuringiensissprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. TheT. nicolonies resistant toBacillus thuringiensisserovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistantT. nistrains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in theT. nipopulations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptibleT. nistrain. The resistance to Cry2Ab in resistantT. niwas an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.

scholarly journals Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

2008 ◽  
Vol 74 (24) ◽  
pp. 7654-7659 ◽  
Author(s):  
Carmen Sara Hernández-Rodríguez ◽  
Adri Van Vliet ◽  
Nadine Bautsoens ◽  
Jeroen Van Rie ◽  
Juan Ferré
Keyword(s):  
Binding Sites ◽  
Mode Of Action ◽  
Helicoverpa Zea ◽  
Tertiary Structure ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Binding Assays ◽  
Cry Toxins ◽  
Long Time ◽  
Competition Assays

ABSTRACT For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

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

2000 ◽  
Vol 66 (4) ◽  
pp. 1553-1558 ◽  
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.

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