ABSTRACT
The widespread and sustainable exploitation of the entomopathogen Bacillus thuringiensis (Bt) in pest control is threatened by the evolution of resistance. Although resistance is often associated with loss of binding of the Bt toxins to the insect midgut cells, other factors have been implicated. Here we used suppressive subtractive hybridization and gene expression suppression to identify additional molecular components involved in Bt-resistance in Plutella xylostella. We isolated transcripts from genes that were differentially expressed in the midgut of larvae from a resistant population, following ingestion of a Bt kurstaki HD1 strain-based commercial formulation (DiPel), and compared with a genetically similar susceptible population. Quantitative real-time polymerase-chain reaction (RT-PCR) analysis confirmed the differential basal expression of a subset of these genes. Gene expression suppression of three of these genes (P. xylostella cyclin-dependent kinase 5 regulatory subunit associated protein 1-like 1, stromal cell-derived factor 2-like 1 and hatching enzyme-like 1) significantly increased the pathogenicity of HD1 to the resistant population. In an attempt to link the multitude of factors reportedly influencing resistance to Bt with the well-characterized loss of toxin binding, we also considered Bt-resistance models in P. xylostella and other insects.
Subject(s)
Bacillus thuringiensis , Biological Control Agents , Insect Proteins/metabolism , Insecticide Resistance/genetics , Moths/metabolism , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endotoxins/genetics , Endotoxins/metabolism , Gastrointestinal Tract/metabolism , Gene Expression , Genes, Insect , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Insect Proteins/genetics , Larva/genetics , Larva/metabolism , Moths/geneticsABSTRACT
The Escherichia coli gene gusA was expressed in the methylotrophic yeast Pichia pastoris in a transcriptional fusion to the homologous methanol-inducible AOX1 promoter. Four recombinant clones were selected for expression studies in shake flask conditions and beta-D-glucuronidase (beta-GUS) activity was assayed each 24 h during the induction period. Regardless of the genomic integration patterns and the gene dosage, beta-GUS was functionally expressed and easily detected in all studied clones. The results obtained demonstrate the feasibility of using this bacterial enzyme as a reporter in Pichia pastoris.
Subject(s)
Escherichia coli/genetics , Genes, Reporter , Glucuronidase/genetics , Glucuronidase/metabolism , Pichia/genetics , Blotting, Southern , Escherichia coli/enzymology , Gene Expression , Mitochondrial Proteins , Oxidoreductases/genetics , Oxidoreductases/metabolism , Phenotype , Pichia/enzymology , Plant Proteins , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Transformation, GeneticABSTRACT
Cry proteins from Bacillus thuringiensis have insecticidal properties. The function of domains I and II has been described but domain III has so far eluded understanding. Domain III from Cry1Ab and Cry1Ac has been cloned, expressed in E. coli and injected to rabbits with the aid of characterizing them immunologically. Interestingly, polyclonal antibodies against Cry1Ab fragment did not recognize either the native Cry1Ab toxin or the Cry1Ac fragment while those against the latter did recognize either the native Cry1Ac toxin or the Cry1Ab protein fragment. A combination of information from sequence comparison and hydrophobicity profile indicates that these protein fragments possibly adopt different spatial dispositions within the respective toxins.
Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Endotoxins/chemistry , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Toxins/genetics , Bacterial Toxins/immunology , Cloning, Molecular , Endotoxins/genetics , Endotoxins/immunology , Enzyme-Linked Immunosorbent Assay , Hemolysin Proteins , Protein Structure, Secondary , Recombinant Proteins/chemistry , SolubilityABSTRACT
Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of delta-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.
