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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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    Optimizing Dothistroma septosporum infection of Pinus radiata and the development of red-band disease : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Barron, Naydene
    The filamentous fungus Dothistroma septosporum infects pine species throughout the world causing red-band disease, one of the most serious diseases of conifer species. In NZ, a clonally derived asexual strain of D. septosporum was identified in 1964, and has spread throughout the country. There are conflicting accounts on the environmental conditions required for infection, which has lead to difficulties in optimizing a laboratory-based system for infection. The pathogen is spread naturally through rain-splashed inoculum of conidiospores from mature stromata that have erupted through the pine needle tissue. Diseased needles become necrotic, often with a red band due to the mycotoxin dothistromin produced by the hyphae. Dothistromin has the chemical structure of a difuranoanthraquinone and shows similarity to the aflatoxin precursor, versicolorin B produced by Aspergillus parasiticus. The role of dothistromin in pathogenicity has not yet been determined, although experiments have shown injecting toxin into pine needles results in the characteristic red band lesion. In this study it was found that fluctuating temperature (16°C/24°C), a 12 h diurnal cycle (white and ultraviolet light), high relative humidity and continuous moisture are conditions conducive to development of red-band disease on inoculated pine trees in an artificial environment. A higher rate of infection was obtained using pine seedlings as opposed to pine cuttings, and using a spore suspension containing a yeast extract. A dothistromin minus mutant was able to infect pine needles, indicating that dothistromin is not a pathogenicity factor, though it may be a virulence factor. The use of GFP-expressing isolates allowed the initial infection process to be monitored with both wild type and mutant isolates. Additionally, a PCR-based diagnostic procedure to confirm infection was developed. The production of aflatoxin by Aspergillus species is regulated by nutritional parameters and extracellular pH, which affect both growth and aflatoxin gene expression. D. septosporum similarly has enhanced growth at acidic pH, but it does not appear that pH has a strong influence on physiological processes as toxin biosynthesis and gene expression do not appear to be pH regulated. Different carbon and nitrogen sources also affect the morphology of D. septosporum.
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    Secondary metabolism of the forest 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, 2016) Ozturk, Ibrahim Kutay
    Dothistroma septosporum is a fungus causing the disease Dothistroma needle blight (DNB) on more than 80 pine species in 76 countries, and causes serious economic losses. A secondary metabolite (SM) dothistromin, produced by D. septosporum, is a virulence factor required for full disease expression but is not needed for the initial formation of disease lesions. Unlike the majority of fungal SMs whose biosynthetic enzyme genes are arranged in a gene cluster, dothistromin genes are dispersed in a fragmented arrangement. Therefore, it was of interest whether D. septosporum has other SMs that are required in the disease process, as well as having SM genes that are clustered as in other fungi. Genome sequencing of D. septosporum revealed that D. septosporum has 11 SM core genes, which is fewer than in closely related species. In this project, gene cluster analyses around the SM core genes were done to assess if there are intact or other fragmented gene clusters. In addition, one of the core SM genes, DsNps3, that was highly expressed at an early stage of plant infection, was knocked out and the phenotype of this mutant was analysed. Then, evolutionary selection pressures on the SM core genes were analysed using the SM core gene sequences across 19 D. septosporum strains from around the world. Finally, phylogenetic analyses on some of the SM core genes were done to find out if these genes have functionally characterised orthologs. Analysis of the ten D. septosporum SM core genes studied in this project showed that two of them were pseudogenes, and five others had very low expression levels in planta. Three of the SM core genes showed high expression levels in planta. These three genes, DsPks1, DsPks2 and DsNps3, were key genes of interest in this project. But despite the different expression levels, evolutionary selection pressure analyses showed that all of the SM core genes apart from the pseudogenes are under negative selection, suggesting that D. septosporum might actively use most of its SMs under certain conditions. In silico predictions based on the amino acid sequences of the proteins encoded by SM core genes and gene cluster analyses showed that four of the SM core genes are predicted to produce known metabolites. These are melanin (DsPks1), cyclosporin (DsNps1), ferricrocin (DsNps2) and cyclopiazonic acid (DsHps1). Gene cluster analyses revealed that at least three of the D. septosporum SMs might be produced by fragmented gene clusters (DsPks1, DsNps1, DsNps2). This suggested that dothistromin might not be the only fragmented SM gene cluster in D. septosporum. According to phylogenetic analyses, some of the D. septosporum SM core genes have no orthologs among its class (Dothideomycetes), suggesting some of the D. septosporum SMs may be unique. One such example is the metabolite produced by DsNps3. Comparison of wild type and ΔDsNps3 D. septosporum strains showed that the ΔDsNps3 strain produces fewer spores, less hyphal surface network at an early stage of plant infection, and lower levels of fungal biomass in disease lesions compared to wild type, suggesting that the DsNps3 SM may be a virulence factor. Attempts to identify a metabolite associated with DsNps3, and to knockout another gene of key interest, DsPks2, for functional characterization were unsuccessful. Further work is required to confirm the gene clusters, characterise the SMs and their roles. However, the findings so far suggest that dothistromin is unlikely to be the only D. septosporum SM that is a virulence factor in since the DsNps3 SM also appears to be involved in virulence. Likewise the fragmented dothistromin cluster may not be the only one in the genome and there may be at least three more fragmented SM gene clusters.
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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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    Investigations of dothistromin gene expression in Dothistroma septosporum and the putative role of dothistromin toxin : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Molecular Biology at Massey University, Palmerston North, New Zealand.
    (Massey University, 2007) Schwelm, Arne
    Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.