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    Genetic analysis of candidate genes that regulate the Epichloë festucae-Lolium perenne mutualistic symbiotic interaction : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Hassing, Berit
    Fungi and plants interact with each other in a multitude of different ways and these interactions can result in different outcomes. The most extreme of these are mutually beneficial interactions, where both partners benefit from the interaction, and pathogenic interactions, where the interaction partners battle each other until one succumbs. For any of these interactions, the fungus relies on two means of communication: (i) it needs to be able to communicate with the host to prevent or elicit defence responses, dependent on the desired outcome, and (ii) it needs to be able to communicate within the hyphal network to ensure nutrient transfer, coordinated growth and development. Here, aspects of these types of communication were analysed in the interaction between the filamentous ascomycete Epichloë festucae and perennial ryegrass (Lolium perenne). Epichloë ssp. are endophytes of cool-season grasses and these interactions are generally mutually beneficial, where the plant benefits through an increased resistance to biotic and abiotic stress, while the fungus feeds on nutrients in the apoplastic space and obtains a means for horizontal and vertical transmission. Recent studies have highlighted the role of fungal small secreted proteins, so called effectors, in preventing host defence responses in pathogenic as well as beneficial plant-fungal interactions. Here, a list of candidate effectors produced by E. festucae was generated and corresponding genes were analysed with regard to genomic location, making use of a fully assembled genome sequence, and expression during growth in axenic culture, in planta and in several symbiosis-deficient mutants. While no association of effector candidate-encoding genes with AT-rich regions, telomeres or clusters was found, they were significantly more likely to contain miniature inverted repeat transposable elements (MITEs) in their promotor sequence. As they were also found to be highly upregulated in planta, it was hypothesised that MITEs are involved in the regulation of effector gene expression in E. festucae. Three candidate effectors and one glycosylphosphatidylinositol (GPI)-anchored protein with a similar expression profile were functionally analysed and found to be secreted. While localisation studies suggested that they remain attached to the fungal cell wall post secretion, genetic deletion or overexpression did not alter the fungus-host interaction, suggesting that these proteins play minor or functionally redundant roles at the observed life stages. Growth of the endophyte in planta is highly regulated and many major signalling pathways are involved in this process, among these signalling via superoxide and other reactive oxygen species (ROS) produced by NADPH oxidase complexes (Nox). Disruption of these pathways results in stunting of the infected host plant and proliferative growth of the fungus. In mammals and plants, lipid signalling is involved in many crucial cellular processes and multiple studies suggest that it is also involved in the regulation of Nox complexes. Therefore, the role of lipid signalling in fungal growth and the interaction with L. perenne was analysed with special focus on a potential role in regulation of the Nox complexes. Of interest here, were the lipid second messengers phosphatidic acid (PA) produced by phospholipase D (PLD), and phosphatidylinositol 4,5-bisphosphate produced by the homolog of the mammalian tumour suppressor protein PTEN. E. festucae generates two structurally different PLDs, and of these, PldB was found to be required for normal hyphal growth and cell-to-cell fusion. Inoculation of pldB deletion strains into L. perenne resulted in severe stunting of the plant accompanied by proliferative growth of the fungus. A PA biosensor was generated and demonstrated a localisation to the cytosol instead of to the plasma membrane as observed in WT. Interestingly, nitroblue tetrazolium (NBT) staining revealed that pldB deletion strains produced less superoxide. Deletion of the PTEN homolog, tepA, did not alter growth of the fungus in culture, but resulted in a mild stunting of the host plant. While lipid biosensors did not localise differently in tepA deletion or overexpression strains, staining of tepA deletion strains with NBT resulted in an increased signal intensity compared to staining of WT. This might indicate that phosphoinositide 3,4,5- trisphosphate, the substrate of TepA, is involved in the regulation of superoxide production. Together, these observations highlight the importance of lipid signalling for fungal growth and mutualistic interactions, and indicate a connection to superoxide production.
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    The role of Epichloë festucae boiA and boiB in symbiotic maintenance of endophyte-grass associations : 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, 2018) Bradley, Ellie Lynn
    The filamentous fungal endophyte Epichloë festucae and its plant host, perennial ryegrass, form a highly regulated mutualistic symbiosis. A key component in the regulation and maintenance of this symbiosis is the production of reactive oxygen species (ROS) by the NADPH oxidase (Nox) complex. Mutations in fungal Nox complex components lead to a disruption in the mutualistic association. In these mutants’ hyphae are often defective in cell-cell fusion and undergo proliferative growth, while the infected plant host is severely stunted. The fungal NoxA complex consists of membrane bound NoxA and NoxD components and cytosolic subunits NoxR, RacA, BemA and Cdc24, which are recruited to the membrane in response to some as yet unidentified signal. This study investigates the hypothesis that BemA is recruited to the membrane via interaction with a BOI1-like protein, known to interact with Bem1 in yeast. E. festucae has two BOI1-like proteins, designated BoiA and BoiB. Phylogenetic analysis suggested the ancestral Boi gene was duplicated in Letiomyceta after the whole genome duplication (WGD) in yeast which resulted in the presence of BOI1 and BOI2. Previous transcriptomic studies have shown boiA is highly expressed in planta compared to in culture and is differentially expressed in mutants that disrupt the mutualistic interaction, suggesting an important role for boiA in symbiotic maintenance. Microscopy analysis showed that in comparison to wild-type, ΔboiA deletion mutants displayed loss of hyphal cell-cell fusion in culture, while in planta ΔboiA mutants exhibited multiple hyphae per intercellular space, intrahyphal hyphae, and hyphal breakage. The resulting ΔboiA infected plants were stunted compared to wild-type infected plants. In contrast, no phenotype was observed for ΔboiB mutants in culture, though in planta intrahyphal hyphae and hyphal breakage was observed. These results suggest that BoiA is required for hyphal cell fusion and regulation of the mutualistic association between E. festucae and perennial ryegrass. While hyphal breakage suggests a role in intercalary growth, the novel mechanism by which E. festucae keeps up with rapid host growth in planta. This study provides an in-depth analysis on the origin and function of E. festucae BoiA and BoiB in culture and in planta.
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    Investigating Epichloë endophyte transmission in Poaceae hosts : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Zhang, Wei
    Vertically-transmitted Epichloë endophytes are agriculturally important fungi that colonise the aerial plant tissues of cool-season grasses within the Poaceae. Plants colonised by selected strains of Epichloë have superior protection from herbivores, thus affirming the important role of these endophytes in New Zealand farming systems. However, the development and marketability of endophyte-based products is often hindered by failures of endophyte transmission. This research investigated: (1) the developmental timing of endophyte colonisation of the seed embryo; (2) the identity of soluble sugars related to endophyte aging during seed storage; (3) the comparative endophyte hyphal density in the shoot apex and florets of high- and low-transmission genotypes; and (4) the molecular mechanisms for endophyte transmission from the inflorescence primordia to the unfertilised ovary. Through a detailed investigation, utilising confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Festuca arundinacea), the endophyte hyphal colonisation in the ovary (pre-fertilisation) through to the fully mature seed stage was tracked. Confocal microscopy images revealed that endophytes have colonised the embryo sac before host grass fertilisation. Tall fescue seeds, either endophyte-free or infected with one of three endophyte strains (AR584, AR605 or common-toxic) were subjected to a 2x2 factorial combination of two factors (accelerated aging or not, and seeds imbibed or not) and the sugar profiles in the seeds were investigated. Trehalose was the sugar that correlated most closely with the loss of endophyte during seed aging. After imbibition, the concentrations of trehalose significantly declined in the endophyte-infected seed tissues, suggesting that the endophyte-oriented trehalose was utilised during imbibition. In addition, the sugar alcohols mannitol and ribitol were found in high concentrations in endophyte-infected embryo and endosperm tissues. These two sugars, therefore, could be potentially used as indexes to estimate endophyte biomass. Two experiments were performed to investigate the endophyte hyphal density in the vegetative and reproductive tissues of perennial ryegrass (Lolium perenne): namely quantification of the endophyte density in the shoot apex tissues using real-time PCR, and analysis of immunoblot colour intensities of laterally bisected florets from six endophyte-grass genotypes (high-transmission [HT]: genotypes 11, 103, 107; low-transmission [LT]: genotypes 13, 79 and 83) and from three positions (bottom, middle and top) of the spike. The florets were collected at three growth stages (Stage I [unfertilised], Stage II [ten days after Stage I] and Stage III [twenty days after Stage I]). Real-time PCR analysis showed that the HT genotypes generally had higher endophyte densities in the shoot apex tissues compared with the LT genotypes. The immunoblot analysis showed that the immunoblot intensities in genotypes 11, 103, 107 and 13 were significantly higher than the other genotypes at Stage I, while the immunoblot intensities in the three HT genotypes were significantly higher than the LT genotypes at Stage II. However, there were no significant differences in the intensities between any of the genotypes at Stage III. Microscopy confirmed that HT genotypes carried a higher density of endophyte hyphae in the shoot apex tissues and ovaries (Stage I) than the LT genotypes. The data indicated that increased endophyte biomass is one factor that enhances endophyte transmission from the parent plant to mature seeds in the HT genotypes. RNA-Seq was used to measure the transcriptional response in two types of tissues (inflorescence primordia and the ovary) in the HT and LT genotypes. This study showed that 102 genes were commonly or exclusively differentially-expressed between the HT and LT genotypes in the inflorescence primordia and/or the ovary. Functional enrichment analyses by agriGO showed that the highly enriched gene ontology (GO) terms between the HT and LT genotypes were involved in serine family amino acid metabolic processes (GO:0009069) and cytoplasmic membrane-bounded vesicle function (GO:0016023) in both the inflorescence primordia and the ovary. More differentially-expressed genes (DEGs) coding for trehalose-6-phosphate phosphatase were induced during development from the inflorescence primordia to the ovary in the HT than the LT genotypes, demonstrating the higher demand for trehalose in the HT than the LT genotypes during endophyte transmission. More genes regulating salicylic acid were significantly repressed while more genes related to jasmonic acid metabolism were significantly induced during development from the inflorescence primordia to the ovary in the HT than the LT genotypes. It is proposed that the lower salicylic acid metabolism and higher jasmonic acid metabolism during development from the inflorescence primordia to the ovary in the HT genotypes might be related to increased endophyte transmission frequencies.
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    The pattern and processes of genome change in endosymbionts old and new : a thesis presented in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Evolutionary Biology, Institute of Molecular BioSciences, Massey University, New Zealand
    (Massey University, 2012) Schönfeld, Barbara Inge Karoline
    Bacterial endosymbionts are an important part of eukaryote evolution as they allow their hosts to exploit bacterial abilities. Plastids, the organelles that enable plant and eukaryotic algae tophotosynthesise are ancient cyanobacterial endosymbionts. Since the initial symbiosis ~1.5 billion years ago the majority of their genes has been lost or transferred to their host’s nucleus. This process has carried on independently in the different lineages following the diversification of the lineage. I have compiled a comprehensive data set of fully sequenced plastid genomes to systematically study the frequency of gene transfers from the plastid to the nucleus across the different lineages.Following the reconstruction of the Plantae phylogenetic tree from plastid encoded proteins, gene loss events were reconstructed along its branches. My calculations show that gene losses have occurred at a relative high frequency and in a lineage specific way. This challenges the original idea that gene transfers from the organelle to the nucleus are rare and chance driven events. Bacteria and eukaryotes continue to form endosymbioses and the study of these relationships produces valuable insights into the early stages of organelle evolution, bacterial metabolic pathways and metabolic regulation. They also allow us a glimpse into the ancient history of eukaryote evolution. For this reason, diatoms that have acquired cyanobacterial endosymbionts with the capability to fix molecular nitrogen were chosen to explore the potential and limitations of high-throughput sequencing technologies for investigating this type of relationship when DNA sequences are obtained from environmental samples and in the presence of bacterial contaminants. The results of this work confirmed the suitability of this relatively new technology to sequence mixed samples but also highlighted i) difficulties in sample preparation which can bias the composition of metagenomic samples obtained, and also ii) the varying suitability of different types of samples used in high-throughput sequencing.