Enhanced toxicity of <i>Bacillus thuringiensis</i> Cry3A δ‐endotoxin in coleopterans by mutagenesis in a receptor binding loop

Wu, Sheng‐Jiun; Koller, Christiane; Miller, Deborah L.; Bauer, Leah S.; Dean, Donald H.

doi:10.1016/s0014-5793(00)01505-2

Cited by 45 publications

(32 citation statements)

References 48 publications

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“…The key role of domain II in three-domain Cry proteins in mediating interactions with insect receptors has been exploited by mutation of amino acid residues in the loop regions of this domain. Mutation of Cry1Ab increased its toxicity toward larvae of gypsy moth (Lymantria dispar) by up to 40-fold (Rajamohan et al, 1996), and similar strategies were used to increase the toxicity of Cry3A protein toward target coleopteran pests (Wu et al, 2000;see Fig. 1).…”

Section: Mutagenesis Of Three-domain Cry Toxinsmentioning

confidence: 99%

Biotechnological Prospects for Engineering Insect-Resistant Plants

Gatehouse

2008

Plant Physiology

194

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Insect-resistant crops have been one of the major successes of applying plant genetic engineering technology to agriculture; cotton (Gossypium hirsutum) resistant to lepidopteran larvae (caterpillars) and maize (Zea mays) resistant to both lepidopteran and coleopteran larvae (rootworms) have become widely used in global agriculture and have led to reductions in pesticide usage and lower production costs (Toenniessen et al., 2003;Brookes and Barfoot, 2005).The source of the insecticidal toxins produced in commercial transgenic plants is the soil bacterium Bacillus thuringiensis (Bt). Bt strains show differing specificities of insecticidal activity toward pests, and constitute a large reservoir of genes encoding insecticidal proteins, which are accumulated in the crystalline inclusion bodies produced by the bacterium on sporulation (Cry proteins, Cyt proteins) or expressed during bacterial growth (Vip proteins). The threedomain Cry proteins have been extensively studied; their mechanism of action involves a proteolytic activation step, which occurs in the insect gut after ingestion, followed by interaction of one or both of domains II and III with ''receptors'' on the surface of cells of the insect gut epithelium. This interaction leads to oligomerization of the protein, and domain I is then responsible for the formation of an open channel through the cell membrane . The resulting ionic leakage destroys the cell, leading to breakdown of the gut, bacterial proliferation, and insect death.However, not all pests are adequately targeted by the Bt toxins used at present, and there is still a need to develop solutions to specific problems, such as resistance to sap-sucking pests and pests of stored products. This Update will review some developments to the basic Bt strategy and selected alternative methods for engineering insect resistance.

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Section: Mutagenesis Of Three-domain Cry Toxinsmentioning

confidence: 99%

Biotechnological Prospects for Engineering Insect-Resistant Plants

Gatehouse

2008

Plant Physiology

194

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“…Also, we tested the theory that surface loops in domain II of Cry 3Aa are involved in receptor binding of P. vorax larvae. Previous results revealed that substitutions within surface exposed loops of domain II on the cry 3Aa can affect reversible and irreversible receptor binding in coleopteran (Wu & Dean 1996, Wu et al 2000. However, none of the mutants was toxic to P. vorax larvae.…”

Section: Discussionmentioning

confidence: 96%

“…Three Cry 3Aa-mutants have been described elsewhere (Wu et al 2000, Wu & Dean 1996 and were constructed by introduction of the mutations D354E (loop 1 in domain II), R345A, ∆Y350, ∆Y351 (loop 1 in domain II), Q482A, S484A and R485A (loop 3 in domain II) in expression plasmid pMH10 derived from pKK233-2 (Pharmacy LKB Biotechnology), using the Kit GeneTailor™ Site-Directed Mutagenesis System (Invitrogen). After mutagenesis, single-strand DNA sequencing was carried out (Macrogen, Korea).…”

Section: Mutagenesis and Protein Isolationmentioning

confidence: 99%

“…Firstly, to characterize a B. thuringiensis native strain collection from P. vorax ecosystem, and secondly, to identify, through bioassays, the biological activity of B. thuringiensis native strains against Andean potato weevil. Also, the toxicity of the Cry 3A δ-endotoxin in P. vorax larvae was evaluated by engineering the specific residues D354, R345, Y350 and Y351 (loop I in the binding domain), and Q482, S484 and R485 (loop III) in domain II (Wu & Dean 1996, Wu et al 2000.…”

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confidence: 99%

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Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

López-Pazos¹,

Martínez²,

Castillo³

et al. 2008

RBT

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Abstract:The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site-directed mutagenesis to modify the Cry3Aa protein.Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with δ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

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“…Mutagenesis and loop swapping experiments with Cry toxins have identified regions of domain II as major determinants of insect specificity (1,51,68,69).…”

Section: Fig 2 Ribbon Representations Of the Cry4aa Fold (A)mentioning

confidence: 99%

Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution

et al. 2006

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The Cry4Aa ␦-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 Å. Like other Cry ␦-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-␣-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a ␤-prism (domain II) and a plant lectin-like ␤-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.

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Enhanced toxicity of Bacillus thuringiensis Cry3A δ‐endotoxin in coleopterans by mutagenesis in a receptor binding loop

Cited by 45 publications

References 48 publications

Biotechnological Prospects for Engineering Insect-Resistant Plants

Biotechnological Prospects for Engineering Insect-Resistant Plants

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution

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