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Item Bioinformatic detection of genetic changes in the fungal endophyte Epichloë festucae AR37 during adaptation to a new perennial ryegrass host : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Microbial Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2020) Razzaq, AsadMutualistic association with the fungus Epichloë festucae var lolii improves the resistance to abiotic stress and herbivory of perennial ryegrass (Lolium perenne). Breeders are interested in moving select E. festucae strains between ryegrass cultivars. In one such attempt E. festucae strain AR37 was transferred from its original ryegrass host to two new ryegrass cultivars. Performance of the resulting novel associations was improved over several years in a breeding program. We wanted to determine if genetic changes in AR37 contributed to this enhanced performance and, if so, identify the nature of these changes. The Epichloë endophyte indeed changed during adaptation to both new host cultivars. We demonstrated this by comparing the genome sequence of AR37 in its original host with pooled “AR37 population genomes” from the two novel associations at the end of the breeding program. These comparisons revealed mutations associated with ~ 150 genes. Frequency of mutations in endophytes increased with the number of seed cycles their new host has gone through. A wide variety of genes including those encoding for certain binding proteins e.g. acting binding, zinc ion binding, DNA-binding, and calcium binding as well as genes encoding for proteins that form signal recognition particles and involved in intracellular signal transduction were amongst those affected by mutations. These genes and their products can play an important role in establishing symbiotic association with the host cultivar. These results indicate that an array of endophyte genes may be involved in establishing a successful association with the new host cultivar. I conclude that (i) the Epichloë genome undergoes functionally relevant alterations as the endophyte adapts to new cultivars and (ii) monitoring the genes encoding the proteins involved, may facilitate breeding programs aimed at improving the performance of new endophyte ryegrass associations.Item Sensing and signalling intercalary growth in Epichloë festucae : 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, 2019) Ozturk, AslinurEpichloë festucae is a seed-transmitted symbiont that colonises the aerial parts of grasses and provides protection from biotic and abiotic stress. Although fungal hyphae normally extend at apical tips, exceptions to polar growth characterise the ecology of many important species. Recently, E. festucae has been shown to undergo intercalary growth during host colonization, where hyphae elongate and new compartments are created between existing compartments. Intercalary growth enables the synchronized growth of E. festucae hyphae and plant cells, and rapid hyphal elongation in plant intercellular tissue. Intercalary growth in E. festucae in vitro has been shown to be stimulated by mechanical stretch, mimicking the forces thought to be imposed on hyphae in plants due to their attachment to growing host cells. This research also showed that the High Affinity Calcium Uptake (HAC) and Cell Wall Integrity (CWI) systems influence intercalary growth, however, the mechanisms that regulate cell wall plasticity and compartmentalization are still largely unknown. The aim of this study was to identify the global gene responses to mechanical stress in E. festucae and to further investigate the roles of HACS and CWI in cell wall plasticity and intercalary growth. First, the role of E. festucae MidA, a homolog of the S. cerevisiae Mid1 stress-activated calcium influx channel complex, was addressed to better understand its involvement in intercalary growth and host colonisation. E. festucae MidA had been partly characterized previously and found to regulate vegetative growth, cell wall morphology, calcium influx and colonisation of the intercalary growth zone of ryegrass leaves. In this study, L. perenne seedlings were inoculated with E. festucae wild type, ΔmidA, and midA complementation strains. The effects of midA deletion on rates of plant colonisation, and the phenotype of infected plants was determined, and found to be the same as the wild type, although hyphae were more difficult to detect. The biomass of the different strains in host tissues, enriched either for the shoot apex (including the meristem) or surrounding leaf tissues, was quantified. The results revealed that E. festucae ΔmidA colonization in both tissue types was reduced compared to wild type (WT), but the effect was most convincing during growth in leaf tissues. These findings suggests that MidA function is required for host colonization, particularly in the leaf expansion zone where intercalary growth occurs. The role of MidA in cell wall plasticity and hyphal growth responses to mechanical stretch was next addressed. E. festucae WT and ΔmidA strains were grown on Potato Dextrose Agar (PDA), with or without 50 mM CaCl2 supplementation in a custom stretching device. When grown on PDA without calcium supplementation, the cell walls of WT and midA-complemented deletion strains were able to withstand mechanical stretching equivalent to 6.5% of their hyphal length (applied over approximately 15 min). However, when grown in the presence of 50 mM CaCl2, wild type hyphae and midA-complemented deletion strains were able to withstand 26% of mechanical stretch without visible evidence of cell wall fracture. In contract, ΔmidA strains grown on PDA alone were damaged after 2% of mechanical stress. Supplemental calcium was able to partly rescue this defect, and the ΔmidA strains were able to undergo 8.9% of stretch in PDA plus 50 mM CaCl2. These findings showed that supplemental calcium increases the resilience of E. festucae cell walls to mechanical stretch, and that midA is required for this, presumably by facilitating calcium influx and cell wall plasticity. Next, a transcriptomics study was conducted on E. festucae cultures undergoing various degrees of stretch. Hyphae were grown in vitro on silicon membranes and stretching forces applied to induce intercalary compartment extension and division, as observed in developing leaves. In cultures harvested 5 min after stretch, 105 genes were differentially expressed, whereas after 3 h, that number increased to 403. Analysis of these genes suggested that reprogramming of primary metabolism and plasma membrane organisation occurs almost immediately in response to mechanical stress, and mobilisation of cell wall enzymes and hyphal growth occurs over a longer time period. Finally, previous research as shown that deletion of E. festucae WscA, a homologue of the S. cerevisiae mechano-sensor Wsc1, induced cell wall and hyphal growth defects during growth in culture, however deletion strains were able to colonise ryegrass plants similarly to the wild type. To further elucidate the role of the CWI pathway in intercalary growth, a comprehensive bioinformatics study was co nducted to identify additional E. festucae Wsc proteins which may function upstream of the CWI pathway. A putative E. festucae WscB homolog was identified, plus a new putative cell wall protein with a unique domain. Phylogenetic analysis showed similar proteins in 17 other Epichloë species and entomopathogenic fungi, suggesting the presence of an E. festucae sensor protein that has been evolutionarily conserved. Vectors to delete these genes were constructed and E. festucae antibiotic-resistant colonies recovered. The putative deletion mutants of both strains were very small and compact compared to wild type growth in culture. Efforts to confirm the deletion loci and functionally characterise the mutants will be part of future research. In conclusion, a transcriptomics study has revealed that mechanical stretching induces metabolic changes during early and late responses in E. festucae, promoting early induction of primary metabolism and later changes associated with hyphal growth and cell wall remodelling. Moreover, further investigation of MidA revealed its importance for cell wall plasticity during intercalary expansion, and indicated that calcium is an essential requirement for hyphal resilience to mechanical stretch. Finally a new protein was discovered that responded to mechanical stress and could be a potential mechanosensor protein. This PhD project attempted to broaden our understanding of intercalary growth in E. festucae and pave the way for future studies on mechanical stress response in fungi.Item 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, BeritFungi 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.Item How does Epichloë festucae avoid the host defence response? : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2018) Noorifar, NazaninEpichloë festucae is a filamentous fungus, which forms symbiotic associations with aerial tissues of Lolium and Festuca grass species. Chitin, a polymer of N-acetyl-Dglucosamine, is an important component of the fungal cell wall and a well-known pathogen associated molecular pattern (PAMP). Chitin promotes pathogen-triggered immunity (PTI) upon hydrolysis with plant chitinases and release of chitin oligomers. Therefore, to establish a stable and successful symbiosis, the endophyte needs to remain ‘hidden’ from the host immune system or actively suppress it. Confocal laser scanning microscopy (CLSM)-based analysis of leaf tissue infected with the E. festucae wild type strain and infiltrated with the chitin-specific molecular probe, WGA-Alexa Fluor-488, showed that only the septa of endophytic hyphae bound this probe while the entire cell wall was labelled in epiphyllous hyphae confirming previous observations that hyphal cell wall chitin is either masked or remodelled in endophytic hyphae. The aims of this project were (i) to test whether E. festucae LysM-containing proteins have a role in binding to or sequestering cell wall chitin oligomers and thereby preventing PAMPtriggered immunity and (ii) to analyse the composition of the cell wall of endophytic and epiphytic hyphae. An analysis of the E. festucae genome identified seven genes encoding proteins with LysM domains. Expression of two of these genes, lymA and lymB, increased in planta compared to in culture. Interestingly, both are divergently transcribed from chitinase encoding genes (chiA and chiB respectively), which also have increased expression in planta. Single gene deletion mutants of lymA, lymB, chiA and chiB as well as a double gene deletion ΔlymA/B were generated, and their plant interaction phenotype analysed. Plants infected with DlymA, DlymB or DchiA had the same plant-interaction phenotype as wild type whereas ΔchiB and ΔlymA/B mutants had defects in hyphal growth within the leaves. Analysis of hyphal cell wall structure using Chitin Binding Protein (CBP) and chitosan (CAP (Chitosan Affinity Protein) and OGA-488)-specific eGFP-based biosensors suggest that cell wall chitin is converted to chitosan in endophytic hyphae. This structural change is consistent with a lack of a defence response when E.festucae forms a mutualistic symbiotic association with L. perenne. Three E. festucae chitin deacetylase genes were identified (cdaA, cdaB and cdaC), and gene expression analysis showed cdaA expression is significantly increased in planta compare to in culture. Functional analysis of cdaA revealed that although plants infected with the ΔcdaA mutant had a similar whole plant interaction phenotype as wild type, they had an abnormal cellular phenotype. Patches of chitin were exposed along the endophytic hyphae confirming this mutant was unable to convert chitin to chitosan. However, hyphae in these plants still labelled with the chitosan biosensor OGA-488 demonstrating that despite the deletion of the cdaA, the hyphal cell wall of endophytic hyphae still contain chitosan suggesting that another chitin deacetylase, possibly CdaB has a redundant function in E.festucae. Collectively these results show that lymA, lymB and chiB are required for establishment of the symbiosis between E.festucae and L. perenne. In addition, this study shows that chitin is converted to chitosan in the hyphal cell wall of endophytic hyphae during the infection and colonisation of the host. The E. festucae chitin deacetylase gene cdaA is also essential for proper hyphal growth in planta and the symbiotic interaction.Item Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2018) Miller, Taryn AngelaAs the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities.Item A study of root aphid Aploneura lentisci Pass. biology and root aphid-host interactions with perennial ryegrass/endophyte associations in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Entomology at Massey University, Manawatū, New Zealand(Massey University, 2019) Müller, Jana LeonieThe root aphid Aploneura lentisci Pass. is an underestimated, under-researched pasture pest likely to become more problematic in New Zealand if the environmental temperature and the frequency of water deficit stress increase, as predicted. The research presented here aimed at gaining first insights into its biology and interaction with plants and endophytes to promote future pest management research. For this purpose, root aphids were observed in model systems (in climate chambers, glasshouse or insectary; in empty microcentrifuge tubes or on diploid perennial ryegrass Lolium perenne L. plants grown on nutrient-enriched agar, with or without endophy Epichloë festucae var. lolii [Latch, M.J. Chr. & Samuels] C.W. Bacon & Schard of the AR1, AR37 or common-toxic CT strains). Apterous neonate offspring, the presumed main dispersal stage of A. lentisci, survived up to four weeks without food (median survival: 8 days). On endophyte-free, mature ryegrass kept at 17 to 21 °C, neonates developed to adults within three to four weeks and lived about two months, feeding mainly on young roots of first and second branching order. Taking into account lower outdoor temperatures, root aphids are thus likely to complete six to nine generations per year in the field. Adults produced 39 to 70 offspring over their lifetime. Presuming a similar nymphal mortality in the field as in the experiments, outdoor root aphid populations could theoretically multiply 23- to 45-fold at each generation. Root aphids raised on endophyte-infected, mature plants were shorter-lived than peers raised on endophyte-free plants. Most aphids on AR37-infected plants did not even reach reproductive maturity. The response to CT-infection was dependent on the plant genotype. Why AR1-infected plants frequently support larger root aphid populations than endophyte-free plants in the field could not be explained by the data collected, however. Root aphid feeding affected the root biomass but not the shoot biomass of perennial ryegrass in the experimental environment. This finding differed from previous reports. Furthermore, colour analyses suggested root aphid feeding could modify some leaf properties. More research will be required to confirm these findings and assess whether irrigation or fertilisation could mitigate root aphid yield losses in the field.Item 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, WeiVertically-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.Item Epigenetic regulation of Epichloë festucae secondary metabolite biosynthesis and symbiotic interaction with Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2017) Lukito, YonathanHistone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require kdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci. Thus, these findings suggest a repressive role for H3K4me3 at these Subtelomeric secondary metabolite loci and are consistent with the role of H3K4me3 in yeast telomeric silencing. Disruption of kdmB did not affect the symbiotic interaction of E. festucae with the host grass but severely reduced the levels of lolitrem B, an animal neurotoxin. At the same time, the levels of ergovaline, another animal toxin, and peramine, an insect feeding deterrent, were not affected. Therefore, disruption or inhibition of KdmB may also serve as a promising approach for future endophyte improvement programmes. The E. festucae homologue of KDM8 (an H3K36me2 demethylase), Jmj4, was further investigated in this study but no H3K6 demethylase activity was found for the protein. Both disruption and overexpression of the gene encoding Jmj4 similarly had no effect on the culture and symbiotic phenotypes of E. festucae. However, deletion of setB, encoding the homologue of yeast Set2 (H3K36 methyltransferase) specifically reduced histone H3K36me3 levels in E. festucae. This contrasts with deletion of Set2 in other fungi which affected H3K36 mono-, di- and trimethylation. The ΔsetB mutant was severely impeded in development, and was unable to establish infection of the host plant. Introduction of the wild-type setB gene reversed these phenotypes. This study shows that H3K4 trimethylation controlled by CclA and KdmB is an important regulator of subtelomeric secondary metabolite genes in E. festucae but not for the symbiotic interaction of the fungus with perennial ryegrass. On the other hand, the histone H3K36 methyltransferase SetB specifically controls H3K36 trimethylation in E. festucae and is required for normal vegetative growth and ability of the fungus to infect the host plant.Item Metabarcoding of the rhizosphere microbiome of perennial ryegrass in response to Epichloë festucae var. lolii infection : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2017) Mahoney-Kurpe, SamEpichloë endophytes inhabit the intercellular spaces of cool-season pasture grasses, and can confer upon their hosts agriculturally desirable benefits such as heightened resistance to biotic and abiotic stresses. The mechanisms underlying many of these benefits are not well understood. Previously observed Epichloë-associated impacts towards the rhizosphere microbiome of their hosts could be a contributing factor, however the overall extent to which specific taxa in the rhizosphere microbiome of perennial ryegrass are affected by Epichloë festucae var. lolii infection remains to be elucidated. To assess this, two independent experiments were carried out in which clonal perennial ryegrass (NuiD) plants inoculated or uninoculated with E. festucae var. lolii (Lp19) originating from sterile tissue culture were grown in soil collected from a natural ryegrass pasture. After approximately two months of growth under controlled conditions in a growth cabinet, their prokaryotic and fungal rhizosphere microbiomes were compared using high-throughput metabarcoding. For prokaryotes, endophyte infection had no significant impact on species richness or evenness of the rhizosphere microbiome of their hosts in either experiment. A very minor but significant shift in overall community composition was shown in the first experiment but not the second. At the level of phyla, aside from a minor 1.1% increase in the relative abundances of Bacteroidetes in the rhizosphere of infected compared with uninfected plants in the first experiment but not the second, there were no other significantly differentially abundant prokaryotic phyla due to endophyte infection. At the genus level rhizospheres of infected and uninfected plants showed a high degree of similarity in both experiments, with little variability between replicates within treatments. At the level of operational taxonomic units (OTUs), in the first experiment there was only one significantly differentially abundant OTU in the rhizosphere depending on endophyte infection, and nine in the second. However, all of which had relatively low abundances (<0.3%), and none were consistently significantly differentially abundant in both experiments. For fungi, there were no significant impacts of endophyte infection on species richness or evenness of the rhizosphere in either experiment, nor were there any significant endophyte-associated shifts detected in overall rhizosphere community composition. Taxonomic analyses found that in both experiments endophyte infected plants had decreased abundances of a single abundant OTU compared with uninfected plants, which was found to be significant across both experiments (P= 0.026). The OTU sequence mapped with moderate (76-90%) homology to a number of reference sequences assigned as belonging to the class Sordariomycetes. Given previously observed endophyte-associated effects on arbuscular mycorrhizal (AM) fungi, reads assigned as belonging to AM were filtered and analysed separately. This showed that there were no significant effects of endophyte infection towards AM diversity nor overall community composition in both experiments, although there was an endophyte-associated increase in the abundance of the AM family Acaulosporaceae in the first experiment but not the second. Thus, aside from an endophyte-associated antagonism towards an abundant OTU in the rhizosphere likely of the class Sordariomycetes, E. festucae var. lolii had an otherwise minor impact on the prokaryotic and fungal rhizosphere microbiome of their perennial ryegrass hosts. The minor magnitude of endophyte-associated effects was further emphasized by analyses consistently showing that both prokaryotic and fungal rhizosphere community composition differed to a greater extent between plants of each experiment irrespective of endophyte infection than between plants of differing endophyte status within each experiment- at least in this cultivar-endophyte strain interaction under the conditions of this study.Item Hordeeae Epichloë endophytes and the formation of synthetic symbioses with cereal grasses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2016) Simpson, Wayne RoydonThis thesis examined two classes of organism that live in symbiosis, grasses and fungi. Specifically it dealt with grasses of the tribe Hordeeae (Triticeae) in the subfamily Pooideae and Epichloë (Epichloë / Neotyphodium) fungi of family Clavicipitaceae. Epichloë endophytes, particularly asexual forms, have important roles in pastoral agricultural systems in the Americas, Australia and New Zealand. Selected strains add value to grass-based forage systems by providing both biotic and abiotic stress resistance. Cereal grasses such as wheat, barley and rye are important to human and animal nutrition and indeed to the foundation and maintenance of Western civilisation. Modern Hordeeae cereal grasses such as wheat, barley and rye do not host Epichloë endophytes, although grasses of some genera within the tribe, such as Elymus and Hordeum, do so. Both organism classes, Epichloë endophytes and cereal grasses, are of great importance in their own contexts; this research examined the possibility of bringing them together in symbiosis with the ultimate goal of improving cereal production systems. In this study, a screen of wild Elymus and Hordeum grasses in Gansu Province, China showed high levels of Epichloë infection. A diverse range of fungal genotypes was identified using SSR markers, and chemical screening revealed the production of alkaloid metabolites consistent with the range seen in Epichloë-infected pasture grasses of tribe Poae. Importantly, strains were identified that did not produce the mammalian toxins ergovaline or Lolitrem B, although less toxic intermediates such as the indole diterpene paspaline and ergot clavine alkaloids were identified. In addition, strains were identified that produced the insect deterrents/toxins peramine and loline. Inoculation studies performed in this study demonstrated that cereal grasses could be successfully infected by artificial means using cultured Epichloë fungus, although infected plants generally had poor morphological phenotypes. While alkaloid production of synthetic associations was qualitatively the same as that of native associations, relative quantitative differences were observed between native Elymus and synthetic rye. Differences in infection frequencies and host phenotypes were observed between Epichloë strains. The choice of Epichloë strain used for inoculation profoundly affected the outcome of the symbiosis, ranging from no infection to stunted plants that died prematurely, infected dwarf plants through to normal phenotype plants. Host genotype was also observed to impact infection frequency and phenotype. Family differences in infection phenotype in outcrossing rye suggested a host genetic basis for the observed variation, while population differences in selfing rye indicated that genetics may not have been the sole driver. Consistent phenotypes were observed from the self-fertilizing cereals wheat and barley but, unlike rye, these were not amenable to recurrent selection. Finally, the infection of wheat alien addition/substitution lines showed that there is potential to select wheat-based germplasm with improved phenotypes. Thus, both Epichloë genotype and host genotype underpinned successful compatible symbiosis. This work demonstrated that cereal grasses could be synthetically infected with Epichloë and that agriculturally useful metabolites were produced by these symbioses. The manifestation of infection phenotypes highlighted the necessity for careful selection of germplasm for inoculation and a need for selection and breeding of cereal grasses after infection.