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    Effector delivery and effector characterisation in Dothistroma needle blight of pines : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Sciences) in Genetics/Molecular Plant Pathology at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Hunziker, Lukas
    The filamentous fungus Dothistroma septosporum causes a serious foliar disease, Dothistroma needle blight (DNB), on Pinus radiata in New Zealand and on many pine species worldwide. Potentially correlated to changes in climate, this disease has been on the rise for 20 to 30 years, and current countermeasures often struggle to contain the damage it causes. A molecular approach to combat DNB could be promising. Effectors are small proteins secreted by pathogens to promote host colonisation, and have been a major focus of plant pathologists in recent years. However, effector biology in pathogens of gymnosperms has received little research attention. Here, candidate effectors (CEs) were selected using a series of computational prediction tools, as well as RNAseq data from a compatible D. septosporum-pine interaction. A shortlist of 55 highly in planta expressed CEs, predicted to be secreted to the apoplast, was characterised in silico. While almost half of them lacked a predicted function, none were exclusive to D. septosporum. Seventeen effector candidates of particular interest were taken forward for functional characterisation. Specifically, these proteins were screened for induction of plant defences in the form of cell death in the model plants Nicotiana benthamiana and N. tabacum using an Agrobacterium transient expression assay. Five CEs induced cell death in these plants, suggesting recognition by the plant defence machinery. Of those five, three are similar to previously described proteins. Effector screening methods are not available for pine, thus various approaches to achieve this were trialled. A high-throughput method to collect each protein in the apoplastic wash fluid of N. benthamiana was developed. This fluid was applied to P. radiata shoots raised from tissue culture to screen for a response, with promising results. Along with an array of D. septosporum CEs, these shoots may ultimately be used to screen for resistant pine genotypes. Selection of genotypes at this early stage could speed up DNB resistance screening for pine breeding and the protocol could be transferred to related pathosystems. This research also contributes to the molecular understanding of forest diseases and effectors that may be common among pathogens of distantly related hosts.
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    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
    (Massey University, 2005) Jin, Hong Ping
    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.
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    Further studies of dothistromin toxin genes in the fungal forest pathogen Dothistroma septosporum : 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, 2007) Feng, Feng, Zhilun
    The fungal pathogen Dothistroma septosporum is the main causal agent of Dothistroma (red-hand) needle blight, which is a devastating foliar disease of a wide range of pine species. Dothistromin is a difuranoanthraquinone toxin produced by D. septosporum and is considered as a possible virulence factor for the disease. Based on the similarity of chemical structure between dothistromin and aflatoxin (AF) /sterigmatocystin (ST) precursors, nine putative dothistromin biosynthetic genes have been identified, which are homologous to their corresponding genes in the AF/ST gene clusters. However, in contrast to all 25 AF biosynthetic genes tightly clustered in one region (70-Kb) of the genome, the dothistromin gene clusters are located on a 1.3-Mb chromosome and separated into three mini-clusters along with non-dothistromin genes. The dotC gene, located in the mini-cluster 1, is predicted to encode a major facilitator superfamily (MFS) membrane transporter involved in secretion of dothistromin. In this work, by constructing DotC-eGFP fusion protein containing mutants, the subcellular localization of the DotC protein was determined to be mainly targeted to the plasma membrane. The biological function of the dotC gene was characterized by targeted gene disruption. The dotC gene disrupted mutants showed a significant reduction of dothistromin production in both the medium and mycelium. In addition, the exponential growth of dotC null mutants was inhibited when exogenous dothistromin was presented and these mutants also displayed more sensitivity than the wild type strain to exogenous dothistromin. The results indicated that the DotC protein is a membrane associated protein and might have a role in dothistromin production and be involved in secretion of exogenously supplied dothistromin toxin. Two novel dothistromin biosynthetic genes, norA/B and verB (partial sequence), were identified by using degenerate PCR and D. septosporum genomic library screening. The putative NorA/B and VerB are postulated to encode a dehydrogenase and a desaturase, respectively and are similar to AF/ST genes. These findings further confirmed that the dothistromin shares biosynthetic pathway steps with AF/ST.
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    Development of a pathogenicity testing system for Dothistroma pini infection of Pinus radiata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 2004) West, Phillip John
    Dothistroma pini is a fungal pathogen of pine species around the world and can be found in most parts of New Zealand. Infection by D. pini causes a disease commonly known as Dothistroma needle blight. Dothistroma needle blight has a significant financial impact on New Zealand's forestry industry. Although control of infection by D. pini is currently very successful there is a possibility that a new strain introduced from another country could be a lot more damaging and overcome current control measures. In recent years both the incidence and severity of the disease have increased in the northern hemisphere and other parts of the world. A distinctive characteristic of Dothistroma needle blight is the production in the infected needle of a toxic red pigment called dothistromin. Dothistromin is produced as a secondary metabolite by D. pini and has known phytotoxic properties as well as clastogenic and mutagenic properties towards human cells. Purified dothistromin toxin injected into pine needles has been shown to reproduce symptoms similar to those observed during D. pini infection. Because of this production, dothistromin is thought to play an important role in the infection process. Mutants of D. pini that are deficient in dothistromin production have been made recently that will allow this role to be investigated. The aim of this study was to develop a pathogenicity testing system under PC2 containment (required for dothistromin deficient mutant) and to develop microscopy methods required to monitor both epiphytic and endophytic growth of the fungus on the needle D. pini requires high light intensity, continuous leaf moisture and a specific temperature range in order to infect pine needles. Progress was made towards developing a robust pathogenicity testing system. This study has also developed several microscopy techniques for the visualisation of epiphytic growth including a fluorescent microscopy technique. Other bright field and fluorescent staining techniques were investigated with some success. Staining techniques were not successful for the visualisation of endophytic D. pini growth but a green fluorescent protein (sgfp) reporter construct was obtained and two gfp plasmid contracts were developed for the transformation of D. pini for use as biomarkers. Successful introduction of the gfp constructs into D. pini will allow in situ visualisation of endophytic and epiphytic D. pini growth. The work done in this study will be useful for the further investigation into the role of dothistromin toxin, which may lead to new or more efficient methods of controlling D. pini as well as possibly providing information about other polyketide molecules of economic or medical significance.
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    Identification and characterization of Dothistroma septosporum effectors : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics
    (Massey University, 2015) Guo, Yanan
    Dothistroma septosporum is the main causal agent of Dothistroma needle blight of pines. However little is known about mechanisms of pine resistance against D. septosporum, or whether there is any classical gene-for-gene resistance involved. The molecular basis of how fungal effector proteins can trigger plant host resistance in a gene-for-gene manner was determined partly by work with the model fungus Cladosporium fulvum and its tomato host. Comparative genome analysis of C. fulvum and D. septosporum genomes identified nine putative effector genes (DsAvr4, DsEcp2-1, DsEcp2-2, DsEcp2-3, DsEcp4, DsEcp5, DsEcp6, DsEcp13 and DsEcp14) in D. septosporum that are homologous to well-characterized C. fulvum effector genes. Other effector candidates were identified as small cysteine rich proteins that are highly expressed in planta, including DsHdp1 which is a hydrophobin gene and Ds69335 which belongs to the sperm-coating protein-like extracellular protein SCP/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) superfamily. Transcriptome analysis showed that, except for DsEcp2-1 and DsEcp6, the in planta expression of D. septosporum effectors was low. Targeted gene replacement of DsAvr4, DsEcp2-1, DsEcp6 and DsHdp1 caused no observed changes in fungal physiology in vitro compared to wild type (WT) and also showed that DsAvr4, DsEcp6 and DsHdp1 are not virulence factors when infecting Pinus radiata. However deletion of DsEcp2-1 caused larger lesions compared to WT, suggesting that DsEcp2-1 may act to suppress a host target which is involved in necrosis induction during the biotrophic infection stage. A domain swap experiment in this study showed that swapping the region between cysteine residues C6 (Cys102) to C7 (Cys114), which contains the chitin binding domain, caused loss of resistance (R) protein Cf-4 recognition of DsAvr4 (with CbAvr4) and gain of Cf-4 recognition of CbAv4 (with DsAvr4 or CfAvr4). Further experiments carried out in Wageningen University showed that a Pro residue located in the chitin binding domain in DsAvr4 is important for Cf-4 recognition, and may have a role in DsAvr4 stability. In this study, effector candidates DsEcp2-1 and DsEcp2-3 were able to trigger a non-host necrotic response in N. tabacum suggesting possible interaction with a N. tabacum protein. Polymorphism analysis showed that DsEcp4 and DsEcp5 have internal stop codons and encode pseudogenes in all the D. septosporum strains tested, except for DsEcp4 in strains from Guatemala and Columbia in which a functional gene is predicted. DsEcp4 and DsEcp5 are the only D. septosporum effectors tested that showed evidence of positive selection. Those results lead to the suggestion that R proteins that recognise DsEcp4 and DsEcp5 may be present in pine species. DsEcp13 appears to be absent from ten D. septosporum strains, suggesting that DsEcp13 is not important for virulence and can also be deleted to avoid an R protein mediated defence response such as a hypersensitive response. Infiltration of DsAvr4, DsEcp2-1 and DsEcp6 P. pastoris expression culture filtrates triggered necrosis in P. radiata needles suggesting that R proteins that directly or indirectly recognise those effectors may also be present in P. radiata. The finding that D. septosporum has homologues of C. fulvum effectors allowed the first study of molecular pathogen-host interactions in this pathosystem. Targeted gene replacement studies identified genes that may have a virulence function and resistance against these effectors may be durable in the field. The pine needle infiltration assay provides a basic screening method to identify pine genotypes that carry resistance proteins and future work in this area is expected to impact on breeding strategies in the forest industry.
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    Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Chettri, Pranav
    Dothistromin is a virulence factor produced by the fungal pine needle pathogen Dothistroma septosporum. It is similar in structure to a precursor of aflatoxin and sterigmatocystin. Unlike most secondary metabolite genes in fungi, the genes for dothistromin biosynthesis are not clustered but spread over six loci on one chromosome. Another characteristic feature of dothistromin synthesis is that dothistromin is produced mainly during the early exponential growth phase in culture. These unusual features have been proposed to be adaptations for the biological role of dothistromin in the disease process. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of aflatoxin and sterigmatocystin in Aspergillus spp. and to address the question of whether genes in a fragmented cluster can be co-regulated. The availability of the D. septosporum genome facilitated identification of orthologs of the aflatoxin pathway regulatory genes aflR, aflJ and the global regulatory genes veA and laeA. These genes were functionally characterised by knockout and complementation assays and the effects of these mutations on the expression of dothistromin genes and the production of dothistromin were assessed. Inactivation of the DsAflR gene (?DsAflR) resulted in a 104 fold reduction in dothistromin production, but some dothistromin was still made. This contrasted with ?AflR mutants in Aspergillus species that produced no aflatoxin. Expression patterns in ?DsAflR mutants helped to predict the complete set of genes involved in dothistromin biosynthesis. AflJ was proposed to act as a transcriptional co-activator of AflR in Aspergillus spp. Disruption of DsAflJ resulted in a significant decrease in dothistromin production and dothistromin gene expression. Interestingly the expression of DsAflR was not affected by deleting DsAflJ, while conversely DsAflJ transcript levels increased significantly in a DsAflR mutant compared to the wild type. Heterologous complementation with A. parasiticus, A. nidulans and C. fulvum AflJ failed to revert the dothistromin level to wild type suggesting species-specific function of AflJ. VeA is an important regulator of secondary metabolism and development in fungi. Inactivation of the D. septosporum ortholog (DsVeA) resulted in reduced dothistromin production and showed the influence of DsVeA on the expression of other secondary metabolite backbone genes. Asexual sporulation was reduced but mutants were not compromised in pathogenicity. Overall, D. septosporum DsVeA showed functional conservation of the usual role in fungi. LaeA is a global regulator of secondary metabolism and morphogenetic development, first identified in Aspergillus nidulans. Unexpectedly, DsLaeA exhibited an unusual repressive function on the dothistromin pathway and DsLaeA mutants exhibited an extended period of dothistromin production compare to WT in vitro. The mutation of DsLaeA showed varied responses in expression of other secondary metabolite genes and had differences in sporulation and hydrophobicity compared to the wild type.
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    Investigation of dothistroma needle blight development on Pinus radiata : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Kabir, Md Shahjahan
    Dothistroma needle blight (DNB), caused by the fungi Dothistroma septosporum and Dothistroma pini, is an important foliar disease of pine species throughout the world and predictions of the future spread of this disease have been made using climate models. Although DNB infection is prevalent in many forests, attempts to achieve infection under controlled laboratory or glasshouse conditions are notoriously difficult. However, artificial infection is a very important tool for studying different aspects of plant-microbe interactions, such as pathogen life style and roles of virulence factors. D. septosporum was thought to have a hemi-biotrophic life style but this was not formally investigated in planta. The non-host selective toxin dothistromin produced by this fungus was shown not to be essential for pathogenicity but its role in pathogen virulence was unknown. The aims of this study were to improve the DNB pathogenicity assay and to use this system to test the hypotheses that D. septosporum is a hemi-biotrophic pathogen and that dothistromin plays a role in virulence. A new sporulation medium (pine needle medium with glucose) was used to obtain sufficient viable D. septosporum spores. The critical microclimatic component of leaf wetness was optimised to have a short (4-7 d) high wetness period followed by 'medium' wetness (continual misting), and using these conditions >80% needle infection was routinely achieved on Pinus radiata seedlings. A combination of microscopy, biochemical and molecular studies over a timecourse of infection of P. radiata by D. septosporum confirmed its hemi-biotrophic life style. Restricted mesophyll colonisation, shorter lesions and fewer spores from P. radiata needles infected with dothistromin-deficient mutants, compared to those with wild type D. septosporum, suggested that dothistromin has a role in virulence. Interestingly ‘green islands’ in which chlorophyll levels were maintained at higher levels than adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wild-type and mutant isolates. At a later developmental stage of the lesion the green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type, which lead to the hypothesis that chloroplasts could be a site of action of dothistromin. The discovery that dothistromin is a virulence factor opens up new insights into the Dothistroma-pine interaction. This fundamental finding will be useful for management strategies for this important disease in the future.
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    Mode of action of dothistromin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, New Zealand
    (Massey University, 1974) Harvey, A M
    Dothistromin is a bright orange-red pigment produced by the pine-needle pathogen Dothistroma pini Hulbary, the causal agent of a necrotic disease known as dothistromal blight. This compound, implicated as a fungal toxin, has been isolated both from laboratory cultures of D. pini and from infected Pinus radiata foliage. Detailed chemical investigation by Gallagher (1971) showed that dothistromin is a tri -α- hydroxyanthraquinone fused to a substituted tetrahydrobifuran ring system. The bifuran ring moiety is incorporated in other fungal metabolites including the extremely toxic and carcinogenic aflatoxin compounds. There is an increasing body of evidence to suggest that these fungal metabolites share a common biosynthetic origin. Bioassay has demonstrated the toxicity of dothistromin to the unicellular green alga, Chlorella pyrenoidosa, and to tissue cultures of P.attexuata. The very low level of solubility of the compound in aqueous solutions has precluded bioassay using pine seedlings. This thesis reports an investigation of the biochemical changes induced by dothistromin in microbiological systems. In the course of this investigation dothistromin has been shown to be toxic to a range of microorganisms in addition to its known toxicity to Chlorella pyrenoidosa. These studies have suggested possible ways of increasing the sensitivity of bioassays for dothistromin. It was found that the addition of dothistromin to liquid cultures of Chlorella as an ethyl acetate solution caused reproducible levels of inhibition, provided that the ethyl acetate concentration was less than 0.5% Batch culture techniques were used to establish the levels of dothistromin required for inhibition of growth of Chlorella. The ratio of dothistromin concentration to cell number was found to be an important factor in the inhibition response. Utilization of synchronous culture techniques permitted the study of biochemical changes induced by dothistromin throughout the cell cycle of Chlorella. Results showed a marked inhibition of the rate of increase of total protein and RNA over the cell cycle with no significant alteration of the rate of DNA increase. A dose-response curve for dothistromin inhibition of growth of Chlorella was established and a more detailed investigation of the action of dothistromin in inhibiting growth was undertaken using radioactive isotopes. By this means it was shown that 3H-uridine and 14C-phenylalanine incorporation into cell material is inhibited within 30 mins of exposure to the toxin. Difficulties encountered in attempts to obtain satisfactory incorporation of label into Chlorella DNA-fractions prevented further investigation of the effect of dothistromin on DNA synthesis in this organism. This led to the investigation of other microorganisms as more suitable experimental systems for this study. Bacillus megaterium KM, proved to be very sensitive to dothistromin and showed rapid incorporation of radioactive isotopes into protein and nucleic acid fractions. Growth curves established that the inhibitory ratios of dothistromin concentration to cell numbers for this organism were in the order of 0.25 μg/cell X 108 (as compared to 2.0 μg/cell X 108 for Chlorella). At this concentration, over the 30 min time course studied, dothistromin had no effect on the incorporation of 3H-thymidine into the DNA fraction. Inhibition of 3H-uridine incorporation was evident at 6 min and very marked by 10 min while the inhibition of 14C-phenylalanine incorporation into protein was not evident until considerably later. The effects of dothistromin in this system were compared with those of antibiotics with known sites of action. Dothistromin inhibition of 3H-uridine incorporation has a similar time course to that shown by actinomycin D, although marked inhibition by actinomycin D is evident at 3 min, whereas dothistromin inhibition is not noticeable until 6 min. On the basis of these results it is suggested that dothistromin interferes with RNA synthesis and that the observed inhibition of protein synthesis is a secondary effect of this impairment. Confirmation of dothistromin action 'in situ'by administration of the compound to pine seedlings is necessary before any definitive statement can be made concerning its role in dothistromal blight. However these results indicate the possible importance of dothistromin in pathology of dothistromal needle blight of pines. Impairment of the RNA synthetic capacity in pine needle tissue by the toxin could rapidly lead to cell death and to the necrosis of needle tissue observed in diseased foliage.
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    Development of methods allowing correlation of Dothistroma and Dothistromin in planta : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2010) Owen, Timothy J.
    Dothistroma septosporum is a fungal pathogen of pines with a worldwide distribution. It is responsible for the disease red band needle blight, in which necrotic lesions appear on infected needles. The red colour of the disease is due to the presence of the mycotoxin dothistromin. This toxin is structurally related to the better characterised mycotoxins aflatoxin and sterigmatocystin. The function of these toxins is unknown, but dothistromin is hypothesised to act as a competition factor. While much work has been done on D. septosporum and dothistromin in broth culture, in planta work has been limited by the methods available. This work focused first on the development of a method for the reliable and high yield extraction of DNA from infected lesions, as previously used methods were found to be inadequate. It was found that the addition of an enzyme lysis step to the Qiagen DNeasy protocol and the replacement of its column purification with chloroform purification gave a greatly increased yield of DNA with an acceptable loss of purity. To allow quantification of dothistromin from the same lesion samples, previously used assay systems were optimised and compared in their accuracy and sensitivity. An HPLC-fluorescence method was found most effective, and was able to accurately quantify dothistromin at single lesion quantities. The developed methods were used to give a correlation between Dothistroma biomass and dothistromin in lesions at various stages of development. While this correlation was not found to be statistically significant, continuation of this work should allow valid conclusions to be drawn. To give insight into the evolution of dothistromin biosynthesis, the genomes of other dothideomycetes were examined for the presence of dothistromin biosynthesis gene homologs. While no homologs were conclusively identified, a number of genes were shown to have similarity to known toxin biosynthesis genes. In summary, while not all research hypotheses were able to be proven or disproven, this work sets a firm basis for future investigation in these areas using the methods developed, and strongly suggests the direction continued study should take.