Further characterisation of the Dothistromin gene cluster of Dothistroma pini : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

The polyketide dothistromin is a toxin produced by the filamentous fungus Dothistroma pini that is thought to play a role in causing Dothistroma needle blight in 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 and also to versicolorin, an intermediate of the well characterised biosynthetic pathways of AF and sterigmatocystin (ST). The structural similarities between AF/ST and dothistromin suggest that genes homologous to AF biosynthetic genes will be involved in dothistromin biosynthesis. AF/ST biosynthetic genes of A. parasiticus and A. nidulans are clustered and hence it is likely that the dothistromin biosynthetic genes are also clustered in a similar manner. Two λ clones, λKSA and λCGV1 containing portions of the putative dothistromin cluster have been isolated in previous studies. Another λ clone λCGV2 was also identified using an aflatoxin gene probe but it is unknown whether it is part of the dothistromin biosynthetic cluster. The λKSA clone contains part of a putative polyketide synthase pks dot (64% identical to A. parasiticus AF biosynthetic gene pksA). Two crucial domains required for functioning are contained within λKSA, the β-keto acyl synthase (KS) and acyl transferase (AT) domains. The putative pks dot is thought be involved in the beginning of the dothistromin biosynthetic pathway, working in a complex with a fatty acid synthase (FAS) to produce the intermediate noranthrone. A gene replacement construct was made using Multisite Gateway TM Recombination, replacing the AT and KS domains with an hph cassette. Disruption of the pks dot gene will confirm it's involvement in dothistromin biosynthesis and could also confirm the role of dothistromin in pathogenicity as if the putative polyketide synthase (pks dot ) is involved in the first step of the dothistromin pathway thus a knockout would form a mutant devoid of any intermediates. Confirming the involvement of pks dot would also provide evidence that like λCGV1, λKSA contains a portion of the dothistromin biosynthetic gene cluster. As the positioning of the three lambda clones λKSA, λCGV1 and λCGV2 relative to one another in the D. pini genome was unknown Southern blot analysis was implemented to identify any relationship between the three lambda clones. No evidence was found to suggest the close linkage of the three lambda clones however this does not discount any linkage at all. Southern blot analysis did provide evidence that ver-2 (77% identity to melanin biosynthetic gene phn1 of Cochliobolus heterostrophus) of λCGV2 is within dose proximity to a putative aflR gene (regulatory gene for activating gene transcription in AF/ST biosynthesis) suggesting a regulatory role of this putative aflR gene in melanin biosynthesis and not dothistromin biosynthesis. Further nucleotide sequencing of the λKSA clone revealed three putative dothistromin genes. Mox dot and ord dot have high amino acid identity to genes involved in the AF/ST pathways (70% identity to moxY and 51% identity to avfA of A. parasiticus respectively), suggesting similar roles in dothistromin biosynthesis. Epox dot showed high amino acid identity to an epoxide hydrolase of A. niger (hyll) suggesting it has a unique role in dothistromin biosynthesis as no homologs are seen in the AF/ST clusters. Southern blotting was also used to confirm the arrangements of genes from the λKSA clone within the D. pini genome. Further characterisation of genes involved in dothistromin biosynthesis will firstly enable understanding of the role of dothistromin in needle blight and secondly will enable further comparative studies between AF/ST and dothistromin.