ABSTRACT
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.
Subject(s)
Bacillus thuringiensis Toxins/genetics , Crops, Agricultural/parasitology , Disease Resistance/genetics , Endotoxins/genetics , Glycine max/parasitology , Hemolysin Proteins/genetics , Tylenchoidea/pathogenicity , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis Toxins/metabolism , Biological Assay , Caenorhabditis elegans , Crops, Agricultural/genetics , Crops, Agricultural/metabolism , Endotoxins/metabolism , Female , Genetic Engineering , Hemolysin Proteins/metabolism , Plant Breeding/methods , Plant Diseases/genetics , Plant Diseases/parasitology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/parasitology , Glycine max/genetics , Glycine max/metabolism , Tylenchoidea/isolation & purificationABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Chromobacterium/chemistry , Coleoptera/genetics , Perforin/chemistry , Amino Acid Sequence/genetics , Animals , Bacterial Proteins/genetics , Complement Membrane Attack Complex/chemistry , Complement Membrane Attack Complex/genetics , Crystallography, X-Ray , Insecticides/chemistry , Models, Molecular , Perforin/genetics , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/genetics , Protein Domains , Protein Structure, TertiaryABSTRACT
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.
