The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.

1 was first discovered in Cecropia silkworm larvae (1), but the functions of this membrane-bound heme protein have been most extensively studied in mammals (2, 3). A microsomal form of cyt b 5 is required for numerous biosynthetic and biotransformation reactions, which include P450-dependent reactions (3), desaturation of fatty acids (4), plasmalogen biosynthesis (5), and cholesterol biosynthesis (6, 7). A soluble form of cyt b 5 is involved in the reduction of methemoglobin in erythrocytes (8) and the biosynthesis of N-glyconeuraminic acid (9). A mitochondrial form, bound to the outer mitochondrial membrane, has been described in mammals as well (10). Cyt b 5 -like sequences are also found as part of larger polypeptides such as flavocytochrome b 2 , sulfite oxidase and nitrate reductase (11), probably as a result of gene fusion events. The role of cyt b 5 in microsomal P450-dependent monooxygenase reactions has been studied most extensively.P450s are a large superfamily of heme proteins which play a crucial role in the biosynthesis of a number of endogenous compounds (steroid hormones, vitamins D 3 , eicosanoids, and so forth) and in the activation or detoxification of a vast variety of xenobiotics. In many of these reactions, cyt b 5 is known to determine the fate of certain substrates by either stimulating (2, 3) or inhibiting (2, 12, 13) substrate metabolism, or even by influencing the type of reaction catalyzed (14). The stimulating effect of cyt b 5 has been thought to result from: 1) enhanced rate of the second electron transfer to P450 (12, 15, 16); 2) increased "coupling" of the reaction, i.e. inhibition of superoxide or hydrogen peroxide formation (2, 12, 17, 18); 3) allosteric effects (19,20); and 4) stimulation of the first electron transfer from P450 reductase to some P450s (21). However, the exact mechanism by which cyt b 5 affects P450-dependent reactions remains unclear.Insect P450s have been extensively studied because of their crucial role in the biosynthesis of hormones regulating insect growth, development, and reproduction (ecdysteroids and juvenile hormones) and in the biotransformation of foreign compounds of synthetic (insecticides) or natural (plant and microbial toxins) origin (22). Metabolism of insecticides by P450s is a major mechanism of insecticide resistance in insects (22, 23), and detoxification of plant toxins by P450s is thought to be an adaptation to the hazards of herbivory (24). Both CYP6A1, an insect P450, which is overproduced in insecticide-resistant strains of the house fly, and NADPH-dependent cytochrome P450 reductase, which provides electrons to P450s from NADPH, have been cloned from the house fly, Musca domestica (25, 26) and expressed in E. coli (27). We have found that epoxidation of the cyclodiene insecticide heptachlor by CYP6A1 is stimulated by rat microsomal cyt b 5 in a reconstituted system.2 Furthermore, immunological evidence for the involvement of cyt b 5 in several P450-dependent monooxygenase activities in house fly microsomes has been reported (28).Our...

No abstract

No abstract

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of trans-regulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alphaE7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation in alphaE7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alphaE7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon.

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b 5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo-and apo-b 5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b 5 . The effects of cytochrome b 5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b 5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.

Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.