Identification of putative dothistromin biosynthetic genes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand
Dothistromin is a polyketide-derived mycotoxin produced by the Pinus pathogen Dothistroma pini, and is thought to be important in the development of the necrotic disease Dothistroma needle blight. Targeted disruption of dothistromin biosynthetic genes will allow the direct assessment of the role of the toxin in D. pint pathogenicity. Dothistromin displays structural and biochemical similarities to the aflatoxins (AF) and sterigmatocystin (ST) which are produced by various Aspergillus species. In our laboratory, knowledge from the well characterised ST/AF pathway is being used to isolate and characterise genes likely to be involved in dothistromin production. The D. pini lambda clone, λCGV1. was isolated from a D. pini genomic library by heterologous hybridisation with a fragment of the Aspergillus parasiticus ver 1 gene (Gillman, 1996). In this study, the complete nucleotide sequence of XCGV1 was determined. Analysis revealed that five genes are located within the 13.3 kb genomic region sequenced Three of these genes (dkr1, dox1 and dte1) display strong similarities to genes contained within the ST/AF biosynthetic gene clusters. The dtp1 gene, located between dox1 and dtp 1, shows similarities to transmembrane efflux pumps and is proposed to be a dothistromin toxin pump. The ddh1 gene, located upstream of dkr1, shows similarities to bacterial dehydrogenases. However, the ddh1 coding sequence contains a premature stop codon (encoding a product of 63 amino acids), indicating that the product may be non-functional. Expression analysis of each gene identified in this study confirmed that dkr1, dox1, dte1 and dtp1 are expressed. However, no obvious expression was detected for the ddh1 gene. Southern blot analysis confirmed the genomic clustering of the genes and indicated that a single copy of each gene was present in the D. pini genome. Due to the biogenetic relationship between dothistromin and ST/AF biosynthesis, and because genes identified in this study show similarities to genes involved in ST/AF production, it is thought that these genes are likely to be involved in dothistromin biosynthesis and constitute part of a dothistromin biosynthetic gene cluster.