Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor (insecticidal crystal
ABSTRACTThe biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryICI were compared between the field population and a laboratory strain. The field population proved resistant (>200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brushborder membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.
We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1 Ab5, an insecticidal crystal protein [ICP] from Bacillus fhuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning o f an aminopeptidase N from the midgut brush-border membrane of Plutrlla xylostella, an insect species of which some populations acquired resistance against Cry1 Ab5. Affinity chromatography on a CrylAb5 matrix was used to isolate a 220-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1 Ab5 and the coleopteran-specific Cry3A d-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sextu midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH,-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdTns)-anchored proteins. Low-stringency hybridization of the tl xylostella midgut cDNA library with M. sexta u p 2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N ( P xylostella Apnl) displays 61 % amino acid identity to M . sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. .xy'lostella Apnl contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P xylostellu apnl gene with PtdIns-specific phospholipase C demonstrated that tl xylostella Apnl is attached to the insect cell tnembrane by a glycosyl-Ptdlns anchor.
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