Molecular Definition of Cytochrome P450 -Mediated Multiple Resistance in Helicoverpa Zea

244 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2003.For four decades, it has been hypothesized that the ability of herbivorous insects to cope with a toxin-rich diet serves as a pre-adaptation for the acquisition of insecticide resistance. To evaluate this hypothesis, I conducted a series of experiments with Helicoverpa zea, a polyphagous noctuid of economic importance. Studies at the population level demonstrated that exposure to xanthotoxin, a linear furanocoumarin, enhanced resistance to the insecticide cypermethrin. Piperonyl butoxide (PBO) synergism and biochemical assays demonstrated that cytochrome P450s are responsible for the documented cross-resistance and that metabolism of these two compounds is mediated by at least one P450. Closely related P450s in the CYP6B subfamily (CYP6B8, CYP6B9, CYP6B27, CYP6B28) are cloned from this species. Phylogenic analysis indicated that these four CYP6B genes represent products of repeated duplication of an ancestral CYP6B gene with recent 5'-polar gene conversions. Analyses of induction patterns indicated that transcriptions of these P450 genes are induced from their basal expression levels in midgut and/or fatbody by the plant defense signaling molecules jasmonate and salicylate, by plant defense end products xanthotoxin, idole-3-carbinol, chlorogenic acid, and flavone, and by a synthetic chemical not naturally encountered, phenobarbital. Molecular modeling indicated that CYP6B8, representative of the four H. zea CYP6B proteins has more flexible overall folding, a larger and more elastic catalytic pocket, and two more substrate access channels than has the CYP6B1 from the specialist Papilio polyxenes. Baculovirus-mediated expression of CYP6B8 and CYP6B1 demonstrates that CYP6B8 is able to metabolize six biosynthetically diverse plant allelochemicals (xanthotoxin, quercetin, flavone, chlorogenic acid, indole-3-carbinol, and rutin) and three insecticides (diazinon, cypermethrin and aldrin), whereas CYP6B1 metabolizes only two of these allelochemicals (xanthotoxin and flavone) and one insecticide (diazinon). These results not only provide compelling genetic and molecular evidence for the ecological/evolutionary association between insecticide resistance and allelochemical adaptation, but also demonstrate that insects arm themselves against subsequent build-up of hostplant toxins by "eavesdropping" on plant defense signals. These results also suggest that, at least in the species examined, counterdefense proteins from generalists are structurally more flexible and functionally more diverse than those from specialists.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD