ABSTRACTMOLECULAR GENETICS OF HISTONE DEACETYLASE INHIBITORS FROM FILAMENTOUS FUNGIByWanessa D. Wight Over the last century, thousands of secondary metabolites from filamentous fungi have been identified. Genes involved in secondary metabolite biosynthesis are often clustered and co-regulated. In this dissertation the genes involved in the biosynthesis of two secondary metabolites, depudecin and HC-toxin, from Alternaria brassicicola and Alternaria jesenskae, respectively, were identified. Both depudecin and HC-toxin are inhibitors of histone deacetylase (HDAC), and HC-toxin is a virulence factor for Cochliobolus carbonum. The gene cluster responsible for depudecin biosynthesis was identified. The cluster has six genes (DEP1-DEP6). Mutation by targeted gene disruption of DEP6, which encodes a predicted transcription factor, demonstrated that the cluster is co-regulated. The polyketide synthase gene encoded by DEP5 was also shown by gene disruption to be essential for depudecin production. Alternaria brassicicola wild type (depudecin +) and the DEP5 knockout mutant (depudecin -) strains were tested for virulence on Arabidopsis thaliana Col-0 and pad3 mutants. Pad3 plants infected with the A. brassicicola depudecin mutant showed a 10% reduction in lesion size, indicating that depudecin plays a minor role in virulence in this pathosystem. Alternaria jesenskae, a newly characterized species, is the first organism other than Cochliobolus carbonum shown to produce HC-toxin. Genome survey sequencing by pyrosequencing of A. jesenskae revealed a series of genes closely related to the HC-toxin biosynthetic genes of C. carbonum. The presence of the HC-toxin gene cluster in both C. carbonum and A. jesenskae allowed us address the comparative evolutionary origins of HC-toxin biosynthesis in A. jesenskae compared to C. carbonum. The high degree of homology of the genes in the two clusters, the presence of multiple copies of each gene in both organisms, and the high conservation of intron/exon structures, indicates a close evolutionary relationship between the HC-toxin clusters in the two fungi. Analysis of a series of housekeeping proteins indicates that the two fungi are phylogenetically closely related. Therefore, evolution of the HC-toxin genes can be explained by evolution from a common ancestor. However, future work is necessary to eliminate the possible role of horizontal gene transfer. A. jesenskae was tested for virulence on maize, cabbage, Arabidopsis thaliana and Fumana procumbens (from which A. jesenskae was originally isolated). No disease occurred on any host, indicating that if HC-toxin has a role in plant pathogenesis that none of these plants are hosts for A. jesenskae, or that HC-toxin has some other role in the biology of A. jesenskae. The results presented in this dissertation indicate that HDAC inhibition by toxins produced by fungal pathogens is critical in some pathosystems but not others. Further work is needed to clearly identify if HDACs play a role in plant defense in dicotyledonous plants such as Arabidopsis.
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