9418155 Chilton The long-term goal of this project is to permit manipulation of genes for biosynthesis of DIMBOA and DIBOA, pesticidal metabolites of maize, to enhance protection of maize and possibly other crops. To undertake such manipulation, all genes in the pathway, especially regulatory genes, will be identified and cloned. Screening of M2 seed from ems-mutagenized pollen has already identified two mutations in enzymes of the pathway and one regulatory gene. The regulatory gene is being tagged by transposon mutagenesis in preparation for cloning. Such a regulatory gene could be of direct use in maize breeding. Screening of ems- mutagenized maize will continue. Mutants, inducers, repressors or varying expression during development will enable isolation of "high DIMBOA" and "low DIMBOA" mRNA to be used in cDNA cloning. Genes encoding at least three steps involving cytochrome P-450 oxygenases will be cloned by a PCR-based strategy using a redundant primer deduced from the conserved haem binding site of P450s. Transposon tagging will also be used to facilitate isolation of genes of the two pathway steps not involving cytochrome P450. Cloning of pathway and regulatory genes will lead to knowledge permitting alteration of expression of genes that affect the level, timing and plant parts in which DIMBOA is produced. This should enable better protection of maize and reduction in use of synthetic pesticides. %%% DIMBOA is a low molecular weight metabolite synthesized at the time of germination of corn seed. It protects the young seedling from predation by most insects, bacteria and fungi. The internal concentration of this natural pesticide decreases in the seedling after the second week so that DIMBOA is no longer capable of deterring attack by predators and pathogens such as corn smut, stalk rot, corn rootworm, European corn borer and corn earworm. It is during the period of declining DIMBOA synthesis in the plant that application of external, Synth etic fungicides and insecticides is required to give high grain yield. This project will identify and isolate the genes controlling biosynthesis of DIBMOA using mutants blocked in various steps of the biosynthetic pathway as well as DIMBOA-deficient mutants and DIMBOA-overproducing mutants. Characterization of the genes controlling DIMBOA synthesis may permit development of strategies to alter the concentration of DIMBOA, the location of DIMBOA within the plant, or the life-stage during which DIMBOA concentration is high in order to better protect the corn crop with its own internal defense, and to reduce the amount of synthetic chemicals sprayed in the field. ***
