Start Date: Jul 15, 2009
End Date: Jul 14, 2014
Phytotoxins that may be involved in the pathogenicity of fungal wilt pathogens that affect cotton yield and quality will be investigated. Specifically, the genes that control their biosynthesis or expression will be identified. The fungal compounds currently being considered for investigation are expected to be derived via a polyketide synthase. This will be confirmed using 13C-labelled feeding studies using singly and doubly labelled acetate under conditions that foster rapid production of the metabolites under investigation. Methods that suppress biosynthesis will also be determined. mRNA will be extracted from mycelium and a pair of degenerate primers corresponding to conserved regions of the ketoacyl synthases (KS) domain of the PKS genes will be used to amplify the KS domain of the PKS genes form the extracted mRNA. The expected base pair will be cloned and transformed into E. coli cells. Plasmids will be isolated from the transformed cells and the inserts sequences will be identified. Once PKS genes are substantiated based on the sequence homology to known fungal KS domain sequences, a pair of primers will be synthesized based on the cloned sequences. mRNA isolated at various incubation periods under both metabolite suppression and induction conditions will be used as a template in RT-PCR to analyze the expression profile of the PKS genes corresponding to the cloned fragments. This will enable us to identify the clones involved in metabolite biosynthesis.