Location: Crop Genetics and Breeding ResearchTitle: Functional characterization of cis-acting elements mediating flavone-inducible expression of CYP321A1) Author
Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/3/2010
Publication Date: 9/18/2010
Citation: Zhang, C., Luo, X., Ni, X., Zhang, Y., Li, X. 2010. Functional characterization of cis-acting elements mediating flavone-inducible expression of CYP321A1. Insect Biochemistry and Molecular Biology. 40:898-908. Interpretive Summary: Detoxification enzymes [such as, Cytochrome P450 monooxygenases (P450s)] play a paramount role in mediating plant-insect interactions. Plants utilize P450s to synthesize toxic defensive compounds to defend themselves against herbivorous insects, whereas insect herbivores employ P450s to detoxify plant defensive compounds they encounter in their host plants. To defer the cost of counterdefense against plant defenses, herbivorous insects have evolved a complex of regulatory machinery to detoxify defensive compounds. However, how plant defensive compounds elicit herbivore counterdefensive cytochrome P450 genes remains largely unknown. To determine the details for defensive compound inducibility of the defensive compound-metabolizing gene (CYP321A1) from the corn earworm, flavones (a group of defensive compounds) were used in this study. A series of molecular studies led to the designation of xenobiotic response element to flavone (XRE-Fla). Further electrophoresis mobility shift assays demonstrated the specific binding site of the XRE-Fla to nuclear extracts from fatbody cells of the corn earworm, and the formation of three specific DNA-protein complexes. These results demonstrated that the basal and flavone-inducible expression of CYP321A1 is regulated primarily by XRE-Fla.
Technical Abstract: How plant defensive allelochemicals elicit herbivore counterdefensive cytochrome P450 genes remains largely unknown. To define the cis-acting elements for flavone inducibility of the allelochemical-metabolizing CYP321A1 from the generalist Helicoverpa zea, functions of varying length of CYP321A1 promoter are examined in homologous H. zea fatbody cells. Progressive 3’ deletions reveal presence of positive cis elements in the 5’ untranslated region (UTR). Progressive 5’ deletions map out one essential element region, four enhancer regions, and two silencer regions. Further fine mapping by progressive 5’deletions localizes the essential basal and flavone inducible element to a 36 bp region from -109 to -74. This 36 bp essential element, designated as xenobiotic response element to flavone (XRE-Fla), contains a 5’ AT-only ATTA inverted repeat (-109 to -100), a GCT mirror repeat (-99 to -93) and a 3’ antioxidant response element-like (ARE-like) element (-92 to -74). Internal deletions and substitution mutations show that both the ARE-like element and the GCT mirror repeat are necessary for the basal and flavone-induced expression, whereas the ATTA inverted repeat is only necessary for the maximal flavone inducibility. Electrophoresis mobility shift assays demonstrate that XRE-Fla specifically binds to nuclear extracts from H. zea fatbody cells via its ARE-like element, forming three specific DNA-protein complexes. These results demonstrate that the basal and flavone-inducible expression of CYP321A1 is regulated primarily by XRE-Fla and secondarily by other cis elements scattered in its promoter and 5’ UTR.