1a. Objectives (from AD-416)
Isolate and identify genes involved in the biosynthesis of phytotoxins involved in pathogenicity of fungal pathogen in cotton.
1b. Approach (from AD-416)
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.
3. Progress Report
Project work in FY 2010 identified potential genes that may be involved in the biosynthesis of phytotoxic compounds produced by Fusarium oxysporum f. sp. vasinfectum. A phylogenetic study has been completed that more clearly delineates the relationship among Fusarium oxysporum races and biotypes. As the work progresses, new information will be developed that will advance our understanding of the pathogenicity of the new types of disease-causing fungus. The ADODR of this project and the cooperator are located in close physical proximity, and are in contact with one another on an ongoing basis. The ADODR and the cooperator (or key personnel working under the cooperator) meet and discuss the direction and progress of the project on a regular basis.