•Bacillus thuringiensis Cry3Aa toxin binds to Colorado potato beetle Prohibitin-1 protein.•The deduced amino acid sequence of CPB Prohibitin-1 cDNA sequence contains cadherin elements.•In the ...presence of anti-PHB-1 antibody, Cry3Aa toxin did not inhibit CPB BBMV cleavage of an ADAM fluorogenic substrate.•Cry3Aa toxic effect was potentiated by Colorado potato beetle Prohibitin-1 silencing.
Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.
Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important ...determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa–ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.
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► An ADAM recognition motif in Bt Cry3Aa toxin domain II is essential for Cry3Aa CPB membrane associated proteolytic processing. ► Cry3Aa toxin membrane interaction through domain II recognition motif is functionally relevant for CPB toxicity. ► Disturbance of membrane integrity affects Cry3Aa toxin membrane associated proteolysis and toxicity against CPB.
Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown thatCry3Aa toxin is proteolytically cleaved by CPBmidgut ...membrane associated metalloproteases and that this cleavage is inhibited by ADAMmetalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAMshedding substrates. In pull‐down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaMin a calcium‐independent manner. Furthermore, we used gel shift assays and 1H NMRspectra to demonstrate that CaMbinds to a 16‐amino acid synthetic peptide corresponding to residues N256‐V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPBmidgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM‐specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.
B
acillus thuringiensis
C
ry3
A
a toxin is a coleopteran specific toxin highly active against
C
olorado
P
otato
B
eetle (
CPB).
We have recently shown that
C
ry3
A
a toxin is proteolytically cleaved ...by
CPB
midgut membrane associated metalloproteases and that this cleavage is inhibited by
ADAM
metalloprotease inhibitors. In the present study, we investigated whether the
C
ry3
A
a toxin is a calmodulin (
C
a
M
) binding protein, as it is the case of several different
ADAM
shedding substrates. In pull‐down assays using agarose beads conjugated with
C
a
M
, we demonstrated that
C
ry3
A
a toxin specifically binds to
C
a
M
in a calcium‐independent manner. Furthermore, we used gel shift assays and
1
H NMR
spectra to demonstrate that
C
a
M
binds to a 16‐amino acid synthetic peptide corresponding to residues
N
256‐
V
271 within the domain
I
of
C
ry3
A
a toxin. Finally, to investigate whether
C
a
M
has any effect on
C
ry3
A
a toxin
CPB
midgut membrane associated proteolysis, cleavage assays were performed in the presence of the
C
a
M
‐specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased
C
ry3
A
a toxin proteolysis and also decreased
C
ry3
A
a larval toxicity.
The mode of action of Cry toxins has been described principally in lepidopteran insects as a multistep process. In this work we describe the mode of action of a Cry toxin active in the common pine ...sawfly
Diprion pini (Hymenoptera, Diprionidae), considered a major forest pest in Europe. Strain PS86Q3 contains a long bipyramidal crystal composed of five major proteins. The N-terminal sequence shows that the 155 kDa protein corresponds to Cry5B toxin and the other proteins belong to the Cry5A subgroup. PCR analysis indicates the presence of
cry5Ac and
cry5Ba genes, suggesting that Cry5A protein should be Cry5Ac. Activation of protoxins with trypsin or with midgut content from
D. pini and
Cephacia abietis (Hymenoptera, Pamphiliidae) (spruce webspinning sawfly), another important hymenopteran forest pest, produced a single 75 kDa toxin that corresponded to Cry5A by N-terminal sequence and is responsible for the insecticidal activity. Homologous competition experiments with
D. pini and
C. abietis brush border membrane vesicles (BBMV) showed that the binding interaction of Cry5A is specific. Membrane potential measurements using a fluorescent dye indicate that Cry5A toxin at nM concentration caused immediate permeability changes in the BBMV isolated from both hymenopteran larvae. The initial response and the sustained permeability change are cationic as previously shown for Cry1 toxins. These results indicate that the hymenopteran specific Cry5A toxin exerts toxicity by a similar mechanism as Cry1 toxins.
Variation of UDE-glucosyltransferase activity, during Drosophila melanogaster development, was analyzed. The endogenous metabolite xanthurenic acid and the xenobiotic compounds 1-naphthol and ...2-naphthol were used as substrates. Developmentally regulated differences were observed for the three substrates, suggesting the presence of UDP-glucosyltransferase isoenzymes. This was further confirmed by FPLC chromatofocusing on a Mono P column: seven peaks of UDP-glucosyltransferase activity (pHs: greater than or equal to 6.3, 5.8, 5.5, 4.9, 4.5, 4.2, less than or equal to 4.0) with either single or overlapping substrate specificity were detected. A single xanthurenic acid:UDP-glucosyltransferase activity (pI 5.8) was found throughout development. In contrast, a gradual increase in the number of 2-napthol:UDP-glucosyltransferase isoenzymes (pI from 6.3 to 4.0) was observed during development, whereas no isoenzymes specific for 1-naphthol were resolved. Based on the distribution and substrate specificity of the eluted peaks in the three developmental stages analyzed, the presence of seven or possibly eight UDP-glucosyltransferase isoenzymes is proposed
Toxicity of Bacillus thuringiensis Cry1B, Cry1C, and Cry1E toxins and B. thuringiensis-based bioinsecticides (Cordalene, Dipel, Foray 48B, and Foray 76B) was investigated in Thaumetopoea pityocampa ...(processionary moth) larvae. Cry1B was toxic, while Cry1C and Cry1E were nontoxic. The toxicity of the bioinsecticides analyzed was of the same magnitude as that of the active purified toxins and determined mainly by one of the toxins present in the bioformulate. We also demonstrated that only the active toxins were able to produce cytotoxic effects on the midgut epithelial cells and specifically bound to T. pityocampa brush border membrane. Analysis of the receptor model of the active toxins showed that Cry1B did not compete for the Cry1A binding site. This finding provides useful information for optimizing the use of B. thuringiensis toxins against T. pityocampa.
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