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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 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 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 Viability of endophytic fungus in different perennial ryegrass (Lolium perenne) varieties kept in different storage conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Manawatū, New Zealand(Massey University, 2017) Kitson, Elizabeth RoseEpichloë endophytes form symbiotic relationships with cool-season grasses of the Pooideae family and are known to synthesise a range of bio-protective alkaloids. These alkaloids can provide the grass host with benefits for greater survival including; deterrence of herbivorous pests, increased persistence, better livestock health and protection from abiotic stressors. The commercialisation of novel endophytes is on the increase, and it is important to ensure the survival of the endophyte is maintained so their benefits can be realised. This study examined the effects of different storage conditions on the viability of three commercial novel endophytes (AR1, AR37 and NEA2/6) and one pre-commercial novel endophyte (815). The different storage conditions were the top of a warehouse, the bottom of a warehouse and a temperature and humidity controlled cool store to simulate current commercial seed storage environments. The viability of different endophytes decreases independently of grass seed germination (p = NS) however there are many factors influencing the endophyte survival. Over the one year storage period there were significant interactions between endophyte x ploidy (host), endophyte x location and endophyte x ploidy (host) x location. The pre-commercial endophyte, 815, had the largest reduction in viable endophyte when stored outside of the controlled cool store dropping 70 percentage points at the top of the warehouse, compared with AR37 (12 percentage points), AR1 (16 percentage points), and NEA2/6 (46 percentage points) (p<.001, LSD = 15.9). In the cool store there was no significant decrease in any of the treatments. As more novel endophyte/grass combinations are released for commercial sale it is important to test each for compatibility and performance post-storage. The results of this study recommend controlled low-temperature, low-humidity storage to maintain endophyte viability. Keywords: Endophyte, Epichloë, perennial ryegrass, Lolium perenne, storageItem The role of the G protein and cAMP/PKA signalling pathway in establishment and maintenance of the mutualistic Epichloë festucae - ryegrass association : 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) Bisson, AlexanderGrowth of the fungal endophyte Epichloë festucae in mutualistic symbiotic association with Lolium perenne (perennial ryegrass) is highly regulated and synchronised with the growth of the host plant leaf. To maintain this pattern of fungal growth in planta, specific signalling between symbiont and its host grass is required. To sense the extracellular environment and respond to changes, filamentous fungi rely on G protein-coupled receptors (GPCRs), which transmit signals predominantly via heterotrimeric G proteins to downstream pathways such as the cAMP/Protein Kinase A (PKA) and MAP kinase signalling pathways. In phytopathogenic fungi, G protein signalling and the associated cAMP/PKA pathways are often essential for a normal host interaction. Signal transduction using the second messenger cAMP to activate the PKA activity is finely balanced through a regulatory feedback loop for signal attenuation regulated by 3’-5’-cyclic nucleotide phosphodiesterases (PDE). Using a blast-based bioinformatic approach, a total of 40 genes encoding putative GPCRs were identified in the genome of E. festucae, grouping into 13 of 14 classes of the recent classification system for fungal GPCRs. Among genes encoding components of the G protein signalling cascade, nine of these GPCRs including the two cAMP receptor-like GPCRs, Gpr1b and Gpr2, show significant transcriptional up-regulation in association with the host compared to the respective expression level when grown in axenic culture. A reverse genetics approach was used to functionally characterise the identified cAMP receptor-like GPCRs (Gpr1a, Gpr1b and Gpr2). While deletion of gpr1a was unsuccessful, plants infected with an E. festucae Δgpr1b mutant showed a severe breakdown of the E. festucae–ryegrass association, whilst no effects were observed for Δgpr2-inoculated plants. Among numerous other genes putatively involved in G-protein and cAMP/PKA signalling, two putative PDEs involved in regulation of the cAMP-mediated signal were also identified. Deletion of pdeH (ΔpdeH), a gene encoding a PDE with high affinity towards cAMP, had a dramatic effect on the endophyte-plant association. In contrast, deletion of pdeL (ΔpdeL), a gene encoding a PDE with low affinity towards cAMP, had no effect on the host interaction phenotype, while primarily modulating the intracellular cAMP level during nutrient-induced activation of the cAMP/PKA ii signalling pathway in axenic culture. Finely balanced cAMP levels are crucial for various cellular processes including hyphal growth, cellular differentiation of asexual development and conidiogenesis. This research identified Gpr1b as an important receptor involved in maintenance of the highly restricted endophytic growth pattern of E. festucae in association with the host, potentially regulated by plant-derived molecules. These signals are mediated by the cAMP/PKA signalling pathway for an appropriate alteration in gene expression and subsequent cellular adaptation. Intracellular cAMP levels triggering these concentration-dependent processes are regulated by PdeH, which specifically acts in planta. This study shows that G-protein and cAMP/PKA signalling play an important role in regulating environmental signalling for establishment and maintenance of the mutualistic association between E. festucae and L. perenne.Item Drought tolerance of perennial ryegrass (Lolium perenne L.) and the role of Epichloë endophyte : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2016) He, LuluPerennial ryegrass is the most important grass species in New Zealand. Due to climate change, drought will become more severe and frequent in New Zealand, which makes it increasingly important to improve drought tolerance of perennial ryegrass. There are many ryegrass cultivars in the seed market; however, very limited information is available about drought tolerance of these cultivars. Therefore, the first aim of this thesis was to compare drought tolerance of several market-leading perennial or long-rotation ryegrass cultivars in order to provide cultivar information for pastoral industry. Epichloë festucae var. lolii fungal endophyte naturally colonises perennial ryegrass. Reported effects of endophyte on drought tolerance of the host perennial ryegrass are multifarious. Therefore, the second aim of this thesis was to investigate effects of endophyte on drought tolerance of perennial ryegrass comprehensively. Two main experiments were conducted in this PhD project. In the first experiment, endophyte-free (E–) and endophyte-infected (E+) cloned plants of seven perennial or long-rotation ryegrass cultivars (Grasslands Commando, Ceres One50, Banquet II, Alto, Bealey, Trojan and Avalon), an un-released elite perennial ryegrass line (URL) and one Mediterranean tall fescue cultivar (Grasslands Flecha) were subjected to a cycle of drought and rehydration from December 2012 to May 2013 while other clones of the same plants were irrigated. In the second experiment, two perennial ryegrass cultivars One50 and Commando infected with and without the AR37 endophyte were subjected to a glasshouse experiment. Eight genotypes of each cultivar with and without endophyte infection were either under irrigation or withheld irrigation for two weeks and then rehydrated for one month. A series of plant morphological and physiological responses were measured in each experiment. In the rainout shelter experiment, it was found that Flecha tall fescue was more tolerant to drought than ryegrass cultivars, but this was attributed to its small plant size induced by the partial summer dormancy. Introducing germplasm from Mediterranean areas would be an option to improve drought tolerance of perennial ryegrass in New Zealand. Among evaluated ryegrass cultivars, Banquet II was relatively more drought tolerant than other cultivars, which was also mainly due to its small plant size. In the glasshouse experiment, it was found that Spanish germplasm based One50 was more drought tolerant than „Mangere? ecotype based Commando, suggesting that Spanish germplasm has conferred enhanced drought tolerance to perennial ryegrass in New Zealand. Under both irrigated and non-irrigated conditions, endophyte infection reduced the herbage yield, decreased the relative water content, osmotic potential and stomatal conductance (as indicated by carbon isotope discrimination) and increased the proline concentration of the host compared to E– plants. Also, a majority of these effects were more pronounced in the URL (infected with AR37) and One50 (infected with AR1). It was concluded that E+ plants are at a disadvantage compared to E- plants when insect pressure is artificially controlled, no matter whether the water availability is high or low.Item Sensing and signalling mechanical stress during intercalary growth in Epichloë grass endophytes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand(Massey University, 2015) Ariyawansa, Kahandawa Geeganaarachchige SameeraEpichloë festucae is an agronomically important seed-transmitted endophytic fungus that grows symbiotically within the intercellular spaces of temperate grass species. This fungus has previously been shown to undergo hyphal intercalary growth during host leaf colonization, a highly unusual mechanism of division and extension in non-apical compartments in vegetative hyphae, as an adaptation to colonise rapidly elongating host cells in the developing leaf. However the exact mechanism that triggers intercalary growth was not known. In this study I aimed to test the hypothesis that intercalary growth is stimulated by mechanical stretch imposed by attachment of hyphae to elongating host cells, and that this stress is sensed by mechano-sensors located on hyphal membranes. To test this hypothesis a novel technique was designed and optimised to stretch fungal hyphae under in vitro conditions. Investigation of un-stretched hyphae showed that de novo compartmentalization occurs in sub-apical compartments of E. festucae hyphae according to a compartment length-dependent hierarchy. Subjecting these sub-apical compartments to mechanical stretching showed that hyphal compartment lengths can be increased while maintaining viability, provided that the stretch is within tolerable limits. It further showed that the stretched compartments undergo de novo compartmentalization (nuclear division and septation) similar to un-stretched hyphae but at a significantly higher rate, fulfilling the basic requirements for intercalary growth. E. festucae WscA and MidA, which are orthologues of a yeast cell wall stress and a stretch-activated calcium channel protein respectively, were functionally characterized in order to test the possible involvement of these mechano-sensors in intercalary ii growth. Their roles in general hyphal apical growth, cell wall construction and integrity maintenance during growth in culture were confirmed. The limited ability of ΔmidA mutants to colonise developing leaves indicated a possible role in intercalary growth, while ΔwscA mutants showed wild-type levels of host colonization. In future, the ΔmidA and ΔwscA mutants will be subjected to mechanical stretch in vitro to further understand their roles in mechano-sensing and intercalary growth. Given the possible involvement of the stretch-activated calcium channel MidA in intercalary growth, a successful technique was developed to study calcium signalling and distribution in E. festucae using the genetically-encoded calcium sensor GCaMP5. Investigations revealed the presence of MidA-driven Ca2+ pulses confined to the hyphal tips with unique signatures of temporal and spatial dynamics generated by influx of Ca2+. The presence of active sub-apical Ca2+ uptake systems were confirmed, manifested as occasional Ca2+ pulses in sub-apical compartments that seemed to increase in frequency with mechanical perturbation, indicating a potential crucial role in mechanical stress-driven intercalary growth. In conclusion a prospective model for intercalary growth in the leaf expansion zone is proposed. Mechanical stretching of hyphae results in increased compartment lengths, accompanied by compartmentalization in sub-apical compartments that allows hyphae to extend along their length. Membrane distortion due to stretching activates MidA, triggering a calcium signalling cascade to stimulate cell wall synthesis and other cellular processes.Item Investigation into the role of PacC in Epichloë festucae development and symbiosis with perennial ryegrass : 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, 2014) Lukito, YonathanIn order to survive and adapt to the environment, it is imperative for fungi to be able to sense and respond to changes in extracellular pH conditions. In ascomycetes, sensing of extracellular pH is mediated by the Pal pathway which is activated by alkaline pH. The signal is subsequently relayed to changes in gene expression by activation of the transcription factor PacC, which is known to regulate various fungal metabolic pathways; including ion tolerance, cell-wall integrity and secondary metabolism. The role of PacC in regulating fungal virulence and pathogenicity has also been studied in several pathogenic fungi, but to date not in a symbiotic fungus. Epichloë festucae is a biotrophic fungal endophyte that forms a stable symbiosis with the perennial ryegrass Lolium perenne. In this mutualistic interaction, secondary metabolites are produced by the fungus that confer bioprotection for the host, and the host in turn provides nutrients and a means of dissemination for the fungus. In this study, deletion (ΔpacC; acid-mimicking) and constitutively active (pacCCA; alkaline-mimicking) mutants were generated to study the cellular roles of PacC in E. festucae. Deletion of pacC resulted in increased sensitivity to salt-stress and reduction in aerial hyphae formation, but did not affect the ability of the mutant to grow under alkaline pH conditions. The pacCCA mutant on the other hand showed greater sensitivity to cell-wall and temperature stresses, and was able to grow under both acidic and alkaline conditions. Aberrant intrahyphal hyphae formation and abnormal conidiation were additionally observed in the pacCCA mutant in culture, but hyphal fusion was unaffected. The expression of secondary metabolite genes both in culture and in planta was largely unaffected in both ΔpacC and pacCCA mutants, and expression analysis of genes known to be pH- and PacC-dependent in other fungi showed that these genes were generally pH- and PacC-independent in E. festucae. The deletion and constitutive active pacC mutants were still able to sense and modify extracellular pH. Deletion of pacC did not affect the endophyte-host interaction, but ryegrass plants infected with the pacCCA mutant were mildly hypertillered. Further examination of the growth of the pacCCA mutant in the plant revealed formation of aberrant convoluted hyphal structures and an increase in hyphal breakage; possible reasons for the observed host plant phenotype. This study provides novel insights into the role of PacC in the agriculturally important endophyte, E. festucae. In this fungus, PacC regulates salt tolerance and cell-wall integrity, but not secondary metabolism and growth at non-neutral pH conditions. The results also show that PacC is involved in the symbiotic interaction between E. festucae and perennial ryegrass but is not crucial in the establishment and maintenance of the symbiosis.Item Molecular and bioinformatic analysis of the perA locus in Epichloë : this thesis is presented as a partial fulfilment of the requirements for the degree of Master of Science (M.Sc.) in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2011) Berry, DanielFungal endophytes of the Epichloë genus form largely mutualistic symbioses with coolseason grasses, systemically colonising the intercellular spaces of the host in a strictly regulated fashion. The endophyte receives protection and sustenance from the host, and in return provides benefits such as increased growth, drought resistance and protection against herbivores. Protection against herbivory is mediated through the production of bio-protective fungal secondary metabolites (SM). Examples of these SMs include lolitrem B, the causative agent of ‘ryegrass staggers’ in stock, and the insect feeding deterrent peramine. The genes responsible for the production of each of these SMs are usually found clustered together in the genome, and are often closely associated with a range of transposon relics. SM gene expression occurs only when the endophyte is growing in planta, indicating the presence of plant-fungal signalling. This study investigated the locus structure and organisation of the gene perA that encodes the non-ribosomal peptide synthetase PerA, which is both essential and sufficient for production of peramine. It was found that perA and its flanking intergenic sequences exhibit considerable transposon-mediated variability across Epichloë, and that this transposon activity is likely responsible for the taxonomically discontinuous production of peramine both within and across Epichloë spp. The major facilitator superfamily transporter gene EF102 is divergently transcribed from and co-regulated with perA (EF103). Transcriptome data were used to identify transcription start sites for both genes. Comparative analysis of the intergenic sequence separating EF102/perA from 10 Epichloë isolates covering six different species refined the perA translation start site, and identified conserved regions in the promoters of both genes proposed to be important for regulation. A motif search identified a conserved DNA motif present multiple times in the promoters of both genes. Deletion analysis of EF102 revealed the gene probably does not encode a peramine transporter, as was hypothesised; however the four independent ΔEF102 mutants exhibited a reduction in peramine production relative to wild type, resulting in an alternative hypothesis that EF102 encodes a transporter for a PerA substrate precursor molecule such as glutamate.