RESUMO
Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.
Assuntos
Toxinas de Bacillus thuringiensis/genética , Produtos Agrícolas/parasitologia , Resistência à Doença/genética , Endotoxinas/genética , Glycine max/parasitologia , Proteínas Hemolisinas/genética , Tylenchoidea/patogenicidade , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Bioensaio , Caenorhabditis elegans , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Endotoxinas/metabolismo , Feminino , Engenharia Genética , Proteínas Hemolisinas/metabolismo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Glycine max/genética , Glycine max/metabolismo , Tylenchoidea/isolamento & purificaçãoRESUMO
The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here ß-tripod. Using mutagenesis, we identified functionally important regions in the ß-tripod domain, which may be involved in target recognition.
Assuntos
Proteínas de Bactérias/química , Chromobacterium/química , Besouros/genética , Perforina/química , Sequência de Aminoácidos/genética , Animais , Proteínas de Bactérias/genética , Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/genética , Cristalografia por Raios X , Inseticidas/química , Modelos Moleculares , Perforina/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Domínios Proteicos , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Glicina/análogos & derivados , Herbicidas , Plantas Geneticamente Modificadas/enzimologia , Zea mays/enzimologia , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/isolamento & purificação , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Resistência a Herbicidas/genética , Cinética , Zea mays/genética , GlifosatoRESUMO
The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.
