Confirmation of the presence of a dothistromin biosynthetic gene cluster in the fungal forest pathogen Dothistroma pini : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

Abstract

The polyketide dothistromin is a toxin produced by the fungus Dothistroma pini and is thought to play a role in causing Dothistroma needle blight on the pine Pinus radiata. Dothistromin is structurally similar to aflatoxin B1 (AF), a highly carcinogenic toxin with no known function, that is produced by the fungus Aspergillus parasiticus. The structural similarities between AF and dothistromin suggest that genes homologous to AF biosynthetic genes found in D. pini are dothistromin biosynthetic genes. AF biosynthetic genes in A. parasiticus and A. flavus are clustered, as are the biosynthetic genes of the structurally similar sterigmatocystin in A. nidulans. Dothistromin biosynthetic genes are also likely to be clustered. Two λ clones, λCGV1 and λBMKSA, containing different portions of this putative dothistromin cluster, have been isolated in previous studies. In this study one gene contained on the clone λCGV1 coding for a putative dothistromin ketoreductase dotA (80.2% identical to A. parasiticus AF biosynthetic gene ver-1) was disrupted with the hygromycin B resistance gene (hph) using targeted disruption via homologous recombination. dotA- mutants were tested for dothistromin production and shown to produce at least 10 - 43 times less than the wild type strain. This confirmed that dotA is involved in dothistromin biosynthesis. Further more, dotA- mutants accumulated the intermediate versicolorin A. This finding provides evidence that λCGV1 contains a portion of the dothistromin biosynthetic gene cluster and the presence of versicolorin A suggests pathway by which dothistromin is synthesised. Other genes homologous to AF and ST biosynthetic genes contained on λCGV1 can now be disrupted in order to determine the extent of the dothistromin biosynthetic cluster on λCGV1. Dothistromin deficient mutants can also be used to determine the role of dothistromin in the pathogenicity of D. pini. Further nucleotide sequencing of the clone λBMKSA revealed the promoter region and the N terminal amino acid encoding sequence of the putative dothistromin polyketide synthase PKSDOT. The partial PKSDOT sequence (amino acids 1-1426) is 62% identical to the A. parasiticus PKSA involved in AF biosynthesis. Preparations to disrupt PKSDOT were made and disruption will confirm its presence on the dothistromin biosynthetic pathway. Sequencing of λBMKSA in this study also revealed a putative dothistromin p450 monooxygenase gene, dcm1, providing more evidence that λBMKSA contains part of the dothistromin pathway. The amino acid sequence of dcml is 59% identical to CYPX from the A. parasiticus AF cluster and 56% identical to STCB from the A. nidulans ST cluster. The function of these homologs has not been ascertained. The discovery of a homolog in D. pini (a species only thought to contain genes for the first part of the AF/ST pathway) provides information about the function of these homologs in AF and ST biosynthesis.