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PubMed2001

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.

ADP Ribose Transferases↗

PubMed2004

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions.

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.

Journal Article↗

PubMed1989

[Proteins and proteinase-resistant polypeptides of delta-endotoxins from Bacillus thuringiensis and their insecticidal activity].

Proteins, solubility in alkaline buffer and proteinase-resistant polypeptides (PRP) of parasporal crystals (delta-endotoxin) from Bacillus thuringiensis were investigated. 19 sorts of crystals were divided into 7 types. Difference of crystalline proteins were coincident with their solubility and PRP. The characters of the protein, solubility and PRP were subspecies-specific basically, but in a few of crystals they were strain-specific. There was a closed relation between the biochemistry property of crystal protein and the toxicity in 2 species of insects. Crystal with protein of MW 130-138 kD kD or MW 130-138 kD and 60-65 kD, and PRP of MW 68-75 kD were high toxic in Bombyx mori (Lepidoptera), but most of them only showed weak or no toxicity in Culex fatigans (Diptera). Crystals containing more than 3 sorts of proteins with MW 15-138 kD in major protein and MW 35-75 kD in minor protein and PRP of 35-65 kD had very strong toxicity in Culex fatigans, but no toxicity in Bombyx mori. The construct of the crystal protein and the importance of the character in protein and PRP for the selection of expected strain and for the work of genetic engineering in Bacillus thuringiensis were discussed.

Animals↗

PubMed1998

Expression and immunolocalisation of the snowdrop lectin, GNA in transgenic rice plants.

Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.

Gene Expression↗

PubMed2005

Insect resistance management in GM crops: past, present and future.

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were first commercialized in 1996 amid concern from some scientists, regulators and environmentalists that the widespread use of Bt crops would inevitably lead to resistance and the loss of a 'public good,' specifically, the susceptibility of insect pests to Bt proteins. Eight years later, Bt corn and cotton have been grown on a cumulative area >80 million ha worldwide. Despite dire predictions to the contrary, resistance to a Bt crop has yet to be documented, suggesting that resistance management strategies have been effective thus far. However, current strategies to delay resistance remain far from ideal. Eight years without resistance provides a timely opportunity for researchers, regulators and industry to reassess the risk of resistance and the most effective strategies to preserve Bt and other novel insect-resistant crops in development.

Animals↗

PubMed2001

Transgenic GNA expressing potato plants augment the beneficial biocontrol of Lacanobia oleracea (Lepidoptera; Noctuidae) by the parasitoid Eulophus pennicornis (Hymenoptera; Eulophidae).

The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F2 progeny produced by parasitoids derived from hosts fed on GNA expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.

Animals↗

PubMed2002

Efficiency of mannose-binding plant lectins in controlling a homopteran insect, the red cotton bug.

Yield losses of different crops due to the attack of various classes of insects are a worldwide problem. Sucking type homopteran pests causing damage to many crop species are not controlled by commonly known insecticidal proteins, namely, Bacillus thuringiensis delta-endotoxin (Bt). This study describes the purification of mannose-binding lectins from three different monocotyledonous plants (Allium sativum, Colocasia esculenta, and Diffenbachia sequina) and their effects on a homopteran insect, the red cotton bug. All of them had a detrimental effect on the growth and development of the insect, where A. sativum bulb lectin showed the highest mortality of all, in particular. The same bulb lectin not only affected the growth and fecundity of the insect but also imparted drastic changes in the color, weight, and size, even on the second generation of the insects which have been reared on artificial diet supplemented with a sublethal dose of the lectin. Thus, this finding opens up a possibility of using this lectin as an important component in crop management.

Animals↗

PubMed1994

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.

Amino Acid Sequence↗

PubMed1998

Cloning and sequencing of cDNA of the insecticidal toxin hirsutellin A

The hyphomycete Hirsutella thompsonii produces an extracellular insecticidal protein, Hirsutellin A. This basic protein, cytolytic against insect cells and capable of inhibiting protein translation, possesses biological features similar to the well-characterized ribosomal-inhibiting proteins (RIPs) alpha-sarcin, mitogellin, and restrictocin. Cloning and DNA sequencing analysis of the 3' and 5' RACE products of HtA cDNA identifies a consensus DNA sequence which encompasses the complete open reading frame of the HtA gene. This gene codes for a precursor of 164 aa which includes a 34-aa leader sequence. The leader sequence of HtA, like those found in RIPs, contains a signal and a pro sequence. The mature 130-aa HtA, having a calculated Mr = 14,159 and pI = 9.21, is considered a stable hydrophilic protein. HtA does not possess the characteristic RNase motif of fungal RIPs but does possess a series of consensus phosphorylation and myristoylation sites and a putative ATP/GTP binding site. The sequence of HtA is unique and does not produce the secondary or tertiary structures characteristic of other fungal RIPs. Copyright 1998 Academic Press.

Journal Article↗

PubMed2000

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella.

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.

Animals↗

PubMed1995

Domain organization of Bacillus thuringiensis CryIIIA delta-endotoxin studied by denaturation in guanidine hydrochloride solutions and limited proteolysis.

Denaturation of Bacillus thuringiensis CryIIIA delta-endotoxin--an insecticidal protein, active against Coleoptera larvae--in concentrated guanidine hydrochloride solutions was pursued by fluorescence and circular dichroism spectroscopy and limited proteolysis. It was found that the protein consists of two fragments that differ by their stability to denaturation by guanidine hydrochloride at pH 3. The less stable fragment corresponds to the N-terminal alpha-helical domain limited by Leu-279; the more stable one starts with Ile-280, contains about 330 amino acid residues, and corresponds to the molecule C-terminal moiety that consist of its two beta-structural domains forming a superdomain.

Amino Acid Sequence↗

PubMed1994

Homology between the seed cytolysin enterolobin and bacterial aerolysins.

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian tree Enterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins from Aeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences of A. hydrophila and A. sobria aerolysins showed several similar regions with many residue identities. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.

Amino Acid Sequence↗

PubMed2002

Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of the European corn borer, Ostrinia nubilalis.

Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis ( Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)). We used an F(2) screen on >1,200 isofemale lines of Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80%. In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.

Alleles↗

PubMed2001

Ion channels induced in planar lipid bilayers by the Bacillus thuringiensis toxin Cry1Aa in the presence of gypsy moth (Lymantria dispar) brush border membrane.

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1-2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P(Cl)/P(NMDG) permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100-500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P(Cl)/P(NMDG) permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.

Animals↗

PubMed2001

Cellular localization and characterization of the Bacillus thuringiensis Orf2 crystallization factor.

In vivo crystallization of the Bacillus thuringiensis insecticidal protein toxin Cry2Aa has been studied in the presence and absence of its associated crystallization factor Orf2. When expressed in the same cell, both proteins were localized to the cuboidal cytoplasmic crystal. When expressed in the absence of Cry2Aa, Orf2 was randomly distributed throughout the cytoplasm. Cry2Aa when expressed in the absence of Orf2 did not form visible crystals but could be recovered from the insoluble fraction of the cell lysate and solubilized at high pH. Purified Orf2 was found to be soluble over a wide range of pH although it could be co-precipitated in the presence of Cry2Aa, suggesting a direct interaction between the two.

Bacillus thuringiensis↗

PubMed2007

A proteinase inhibitor from Nicotiana alata inhibits the normal development of light-brown apple moth, Epiphyas postvittana in transgenic apple plants.

Insecticidal proteins are a potential resource to enhance resistance to insect pests in transgenic plants. Here, we describe the generation and analysis of the apple cultivar 'Royal Gala' transgenic for Nicotiana alata (N. alata) proteinase inhibitor (PI) and the impact of this PI on the growth and development of the Epiphyas postvittiana (light-brown apple moth). A cDNA clone encoding a proteinase inhibitor precursor from N. alata (Na-PI) under the control of either a double 35S promoter or a promoter from a ribulose-1,5-bisphosphate carboxylase small sub-unit gene (rbcS-E9 promoter) was stably incorporated into 'Royal Gala' apple using Agrobacterium-mediated transformation. A 40.3 kDa Na-PI precursor protein was expressed and correctly processed into 6-kDa proteinase inhibitors in the leaves of transgenic apple lines. The 6-kDa polypeptides accumulated to levels of 0.05 and 0.1% of the total soluble protein under the control of the rbc-E9 promoter and the double 35S promoter, respectively. Light-brown apple moth larvae fed with apple leaves expressing Na-PI had significantly reduced body weight after 7 days of feeding and female pupae were 19-28% smaller than controls. In addition, morphological changes such as pupal cases attached to the wing, deformed wings, deformed body shape, and pupal cases and curled wings attached to a deformed body were observed in adults that developed from larvae fed with apple leaves expressing Na-PI, when compared to larvae fed with the non-transformed apple leaves.

Animals↗

PubMed1997

Southern analysis of BT-R1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis.

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.

Animals↗

PubMed2005

Effects of Bt maize on the herbivore Spodoptera littoralis (Lepidoptera: Noctuidae) and the parasitoid Cotesia marginiventris (Hymenoptera: Braconidae).

Recent studies have shown that transgenic insect resistant plants can have negative effects on non-target herbivores as well as on beneficial insects. The study of tritrophic interactions gives insight into the complex mechanisms of food webs in the field and can easily be incorporated into a tiered risk assessment framework. We investigated the effects of transgenic maize (Zea mays) expressing insecticidal proteins derived from Bacillus thuringiensis (Bt maize) on Spodoptera littoralis, a non-target herbivore, and on the hymenopteran parasitoid Cotesia marginiventris. In a laboratory study, S. littoralis larvae were reared for their whole lifespan on a mixture of leaves and stems from 2-4-week old Bt maize plants. S. littoralis survival, developmental times and larval weights were significantly affected by Bt maize diet. However, adult moths, which survived development on Bt maize, were the same size as the adults from the control group. C. marginiventris survival, developmental times and cocoon weights were significantly negatively affected if their S. littoralis host larva had been fed Bt maize. ELISA tests confirmed that S. littoralis larvae ingest high amounts of CrylA(b) toxin while feeding on Bt maize. In S. littoralis pupae and in C. marginiventris cocoon silk, only traces of the toxin could be detected. No toxin was found in S. littoralis and C. marginiventris adults. Thus the toxin is not accumulating in the trophic levels and in fact appears to be excreted. Our results suggest that the effects on C. marginiventris when developing in susceptible S. littoralis larvae are indirect (host mediated). The biological relevance of those results and the significance of this study in risk assessment are discussed.

Animals↗