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Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentida…
2014
ABSTRACT Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas , 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 in Cylas puncticollis to 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-…
Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies
2004
Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua,…
Immunohistochemical Detection of Binding of Cryia Crystal Proteins of Bacillus thuringiensis in Highly Resistant Strains of Plutella xylostella (L.) …
1995
We detected binding of insecticidal crystal proteins from Bacillus thuringiensis in one susceptible strain and six resistant strains of diamondback moth, Plutella xylostella, from Hawaii. Immunohistochemical tests with tissue sections from larval midguts showed specific binding of CryIA(a), CryIA(b), and CryIA(c) to brush border membranes. CryIE, which is not toxic to P. xylostella, did not bind to midgut tissues. Larvae from one of the resistant strains ingested extremely high concentrations of a commercial formulation containing the three CryIA proteins without suffering midgut cell damage or mortality. This same resistant strain had previously been found to have greatly reduced binding o…
Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dis…
2007
Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with lab…
Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella)
2003
Abstract Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton. The most common mechanism of insect resistance to B. thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut. We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide. Using Cry1Ab and Cry1Ac labeled with 125I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca. In the susceptible strain, Cry1Aa, Cry1Ab, Cry1…
Bacillus thuringiensis crystal proteins CRY1Ab and CRY1Fa share a high affinity binding site in Plutella xylostella (L.).
1996
The future success of Bacillus thuringiensis based insecticides depends in part on our ability to prevent insects from developing resistance against their insecticidal crystal proteins. Two recent papers indicated cross-resistance between Cry1A proteins and Cry1Fa in two different insect species (Tabashnik et al., 1994, Appl. Environ. Microbiol. 60, 4627-4629; Gould et al., 1995, J. Econ. Entomol. 88, 1545-1559). Brush border membrane vesicles were prepared from Plutella xylostella and used in binding assays with 125I-labeled trypsin-activated crystal proteins. Competition experiments showed that Cry1Fa competed with Cry1Ab for a same binding site, though the latter still bound to a differe…
Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C
2000
ABSTRACT A field-collected colony of the diamondback moth, Plutella xylostella , had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis . After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous…
The CFTR associated protein CAP70 interacts with the apical Cl-/HCO3- exchanger DRA in rabbit small intestinal mucosa.
2005
DRA (down regulated in adenoma) is an intestinal anion exchanger, acting in parallel with NHE3 to facilitate ileal and colonic NaCl absorption. Furthermore it is involved in small intestinal bicarbonate secretion. Because DRA has a PDZ interaction motif, which may influence its properties, we searched for DRA-interacting PDZ adapter proteins in the small intestine. Using an overlay assay with the recombinant DRA C-terminus as a ligand, a 70 kDa protein was labeled, which was restricted to the brush border membrane in rabbit duodenal and ileal mucosa and was not detected in the colon. Destruction of the C-terminal PDZ interaction motif abolished this band, suggesting a specific protein-prote…
Decrease of mRNA levels and biosynthesis of sucrase-isomaltase but not dipeptidylpeptidase IV in forskolin or monensin-treated Caco-2 cells.
1991
International audience; Treatment for 48 h of differentiated, confluent Caco-2 cells with 2.5 10(-5) M forskolin or 10(-6) M monensin, which produces a significant decrease of the de novo biosynthesis of sucrase-isomaltase, does not change quantitatively the de novo biosynthesis of dipeptidylpeptidase IV. Western blot analysis and silver nitrate staining indicate that neither drug induces any modification in the steady state expression of these two brush border hydrolases. Northern blot analysis shows that the level of dipeptidylpeptidase IV mRNA does not change in treated as compared to control Caco-2 cells. In contrast, forskolin and monensin dramatically decrease the level of sucrase-iso…
Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.
2012
ABSTRACT Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differe…