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    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
    (Massey University, 2004) Teddy, Olivia Rachel
    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.
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    Identification of dothistromin biosynthetic pathway genes : 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
    (Massey University, 1996) Gillman, Carmel Jane
    Dothistromin is a polyketide-derived toxic secondary metabolite produced by the filamentous fungus Dothistroma pini which causes the disease Dothistroma needle blight in Pinus radiata. Dothistromin is considered to be an important component in the disease process, although its exact function is yet to be identified. By isolating and identifying genes involved in dothistromin biosynthesis, and subsequently obtaining mutants blocked or altered in the synthesis of dothistromin, the role of this toxin in pathogenicity will be able to be assessed. Dothistromin is structurally related to the mycotoxins, aflatoxin (AF) from Aspergillus parasiticus and A. flavus, and sterigmatocystin (ST) from A. nidulans. Three intermediates in the ST and AF biosynthetic pathways (averantin, averufin, and versicolorin B) are thought to also be intermediates dothistromin biosynthesis. Due to these similarities, cloned AF pathway genes were used as heterologous probes in Southern hybridisation analysis to provide a direct method for identifying dothistromin biosynthetic genes. A fragment of the A. parasiticus nor-1 gene, encoding a reductase involved in the conversion of norsolorinic acid (NA) to averantin (AVN) in the AF biosynthetic pathway, was used as a probe to detect a region of sequence similarity to D. pini genomic DNA. A D. pini genomic library was then constructed and screened, resulting in clone λCGN2. However, Southern hybridisation analysis suggested that this clone did not contain a homologue of the nor-1 gene from A. parasiticus. A fragment of the Aspergillus parasiticus ver-1 gene, encoding a reductase involved in the conversion of versicolorin A (VA) to ST in the AF biosynthetic pathway, was also used as a probe to detect a region of sequence similarity to D. pini genomic DNA. The D. pini genomic library was then screened. Two clones, λCGV1 and λCGV2, were isolated and Southern hybridisation analysis confirmed that these clones contained sequences hybridising to the A. parasiticus ver-1 gene fragment. Fragments of these clones which hybridised were then sequenced and compared to the GenBank database. The amino acid coding sequence of a 0.8 kb SalI region from clone λCGV1 exhibited a high degree of similarity with the A. nidulans verA and A. parasiticus ver-1 genes, involved in the ST and AF biosynthetic pathways, and the Magnaporthe grisea ThnR, and Colletotrichum lagenarium Thr1 genes, involved in melanin biosynthesis. This data suggested a ver-1 homologue is present in the D. pini genome. Limited sequence analysis of a 2.1 kb region from clone λCGV2 suggested that a second independent copy of a ver-1-like gene may also be present in the genome.
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    Characterisation of a putative dothistromin biosynthetic cluster : 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
    (Massey University, 1999) Laarakkers, Seth
    The fungus Dothistroma pini is a key pathogen in New Zealand (and international) softwood plantations, most notably P. radiata. The mycotoxin dothistromin produced by this saprophytic fungus is believed to play a major role in its pathogenesis. Dothistromin shares functional groups and pathway intermediates with those of sterigmatocystin and aflatoxin, secondary metabolites of Aspergillus sp. As the sterigmatocystin and aflatoxin biosynthetic pathways are characterised this provided us with a model pathway and potential probes for the isolation of dothistromin genes. The verl gene is critical to the completion of aflatoxin biosynthesis in Aspergillus sp. as its disruption prevented the synthesis of aflatoxin. Assuming similar enzymes act in the dothistromin biosynthetic pathway a probe for ver1 was obtained and used to probe a D. pini genomic library. This led to the isolation of two lambda clones named λCGV1 and λCGV2 (Gillman 1996). A second library screen was completed using an aflatoxin polyketide synthase (PKS) probe and led to the isolation of the lambda clone λBMKSA (Morgan 1997). The λCGV1 clone has been studied in detail and shown to contain a gene similar to aflatoxin ver1 (named dkr1) and other potential dothistromin biosynthetic genes (Monahan 1998). This study looks in greater detail at the lambda clones λCGV2 and λBMKSA and determines whether they contain putative dothistromin biosynthetic genes and are part of the anticipated gene cluster. In this project the lambda clone λCGV2 was partially characterised which revealed that the other potential ver gene showed a greater similarity to the melanin biosynthetic gene phn than to the aflatoxin gene ver-1. This implied that the clone was unlikely to contain dothistromin biosynthetic genes so no further sequence was generated. However, a partial restriction map was constructed. The other lambda clone, λBMKSA was then further characterised. Double stranded sequence of the putative pks gene region was completed. The remainder of the lambda clone was subcloned and exploratory sequence revealed a gene with high similarity to stcW. The next stage was to determine how the three lambda clones were related. This was approached by probing genomic Southern blots with the ends of the lambda clones to determine the presence of commonly hybridised fragments. The presence of common fragments suggests that the three clones are very close together in the genome, although the evidence which links λCGV2 and λBMKSA is stronger than the evidence that links λCGV2 and λCGV1. This is the first evidence that the three lambda clones isolated using aflatoxin probes are close together in the genome of D. pini. The genes present on these lambda clones show a high degree of similarity to their aflatoxin counterparts and could potentially contain a dothistriomin biosynthetic cluster.
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    Characterisation of a global collection of Dothistroma pini isolates : 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
    (Massey University, 2000) Ganley, Rebecca Jayne
    Dothistroma pini is a filamentous fungus which infects Pinus radiata, New Zealand's predominant forest species. Dothistroma blight causes premature defoliation, a reduction in the rate of growth and, in extreme cases, death of the trees. This forest pathogen produces a toxin, dothistromin, which is implicated in the development of the disease symptoms. Only one strain of D. pini is thought to be present in New Zealand. However, world-wide there is a diverse range including the sexual form. A collection of D. pini strains from eight countries was collated in the UK. To prevent further introductions of 'foreign' D. pini to New Zealand and to assist in the identification and appropriate containment, should a new outbreak of needle blight occur, the D. pini isolates in this collection were characterised at both the species and individual strain level. Sequence analysis of the ribosomal internal transcribed spacer (ITS) and the production of dothistromin by the isolates in the collection confirmed all were D. pini. Quantification of the levels of dothistromin produced by the isolates, in culture, showed a large variation between the strains. Isolates MINI11, NEB8, GUA1 and, in particular, ALP3 produced significantly more dothistromin than NZE1. Changes in culture environment and media types were shown to affect the levels of dothistromin produced by the D. pini isolates. However, these changes were not sufficient to support the production of aflatoxin. To analyse the genetic diversity among the overseas D. pini isolates, a robust microsatellite-based DNA fingerprinting system was developed. Microsatellite loci were isolated. Primers designed to flank the microsatellite repeats were used for PCR amplification in the 'core' twelve D. pini strains. The unique fingerprint patterns obtained from these loci were used to distinguish the isolates to the individual strain level. This system of identification provides an effective tool for screening and prognosis of infected pine forest sites.