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:
The goal of this project is to determine what metabolic or genetic factors control the production of compounds toxic to plants (i.e., phytotoxins) that are produced by pathogenic fungi. The focus is on the fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov). Previous work by this project established that fusaric acid is an important virulence factor for the root-rotting group of Fov isolates like California race 4. Work during FY 2013 developed a reliable bioassay to determine what Fov isolates are most pathogenetic to cotton. Using this method we established that fusaric acid added to the growth medium significantly increased the virulence of the fusaric acid non-producing Fov isolates. That is, even a weakly pathogenic Fov caused severe disease in cotton when plants are first treated with fusaric acid. This has confirmed that fusaric acid is a critical factor in the wilt disease by root-rotting Fov isolates like race 4 that are prolific producers of fusaric acid. As the work progresses, new information will be developed that will advance our understanding of the pathogenicity of these new types of disease-causing fungi. Ongoing work will provide foundational information to support efforts aimed at developing new disease-resistant cotton cultivars for productive use by U.S. farmers.