Molecular analysis of secondary metabolite formation in Alternaria alternata with a special focus on perylene quinones (PQs)

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Fungi of the genus Alternaria belong to the “black molds” and may infect and colonize a variety of agricultural crops such as tomatoes and cereals. Several secondary metabolites, such as alternariol (AOH) and its derivatives can have serious effects on human and animal health and are therefore classified as mycotoxins. Perylenequinones (PQs), such as altertoxin I-III (ATX) are other examples of Alternaria toxins. The genome of A. alternata contains a number of secondary metabolite gene clusters, where it is unknown for which molecule they are responsible. The proposed project aims at the characterization of secondary metabolite genes, especially for the PQs altertoxins I-III, and their regulation. In preliminary experiments it was found that PQ and melanin biosynthesis share the initial polyketide synthesis. It appears that only a few genes necessary for PQ production are clustered together with the polyketide synthase gene, pksA. In order to identify other PQ genes, a reverse genetics approach will be followed, because ATXs biosynthesis resembles cercosporin biosynthesis in Cercospora beticola, where some genes are known. In addition, the Alternaria genome sequences deposited in the databases will be analyzed for further candidate genes. It is possible that in some isolates the genes are clustered. Furthermore, RNAseq analyses of A. alternata grown under conditions of high PQ biosynthesis will be performed and compared with conditions of low PQ production. Mutant strains of the corresponding candidate genes will be generated and analyzed for PQ and the intermediates. This will allow us to propose the biosynthetic pathway for PQs in A. alternata. As a second objective, we will study the regulation of PQ gene expression. The role of PQs in virulence will be tested with tomatoes. Finally, five remaining gene clusters will be studied to an extend to at least identify the corresponding molecule for which biosynthesis they are responsible.