Further characterization of Dothistromin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Abstract

Dothistroma septosporum is a forest pathogen that causes a disease called Dothistroma needle blight. The symptoms are thought to be due to the accumulation of dothistromin toxin produced by D. septosporum. Dothistromin is characterized as a difuranoanthraquinone and shows remarkable similarity to the aflatoxin (AF) and sterigmatocystin (ST) precursor versicolorin B. The similar structure to AF/ST suggests that dothistromin biosynthesis shares biosynthetic steps with the AF/ST pathway. The AF gene cluster in Aspergillus parasiticus and ST gene cluster in A. nidulans have been well characterized. Nine putative dothistromin biosynthetic genes have been identified. One of them, dotA was previously characterized by gene disruption and shown to have a similar function to homologous genes in AF/ST biosynthesis. Two additional putative dothistromin biosynthetic genes, pksA and epoA, were characterized by gene disruption in this study. The inability of the pksA mutants to produce dothistromin indicated that the pksA is a key gene in dothistromin biosynthesis. The feeding of intermediates confirmed that pksA gene product is required for a very early step of dothistromin biosynthesis. The pksA mutants also showed reduced sporulation compared to wildtype, suggesting a relationship between dothistromin production and sporulation. The epoA gene replacements were also obtained successfully by homologous recombination. Both Southern blot and northern hybridization confirmed that the epoA gene was disrupted. However, the epoA mutants did not show any difference to the wild type in three analyses (growth rate, sporulation rate, dothistromin biosynthesis). However it was not possible to rule out a role for EpoA at a very late stage of dothistromin biosynthesis. RACE analysis of the nine identified dothistromin genes characterized the transcription start and stop sites of the genes. Analyzing the putative regulatory protein binding motifs in the untranscribed region of the genes provided clues about the regulation of dothistromin biosynthesis and suggested there might be an aflR-like gene that governs dothistromin biosynthesis. Both the pksA gene disruption and the RACE results suggested that the dothistromin biosynthetic pathway is homologous to that of AF/ST biosynthesis. Further work on the dothistromin gene cluster will help us to understand the evolution of fungal toxin gene clusters