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Item Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.(Frontiers Media S.A., 2023-11-13) Forte FP; Malinowska M; Nagy I; Schmid J; Dijkwel P; Hume DE; Johnson RD; Simpson WR; Asp T; Morillas JIVEpichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.Item Corrigendum: Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.(Frontiers Media S.A., 2023-11-13) Forte FP; Malinowska M; Nagy I; Schmid J; Dijkwel P; Hume DE; Johnson RD; Simpson WR; Asp T[This corrects the article DOI: 10.3389/fpls.2023.1258100.]. This article is a correction to: Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37Item Leaf regrowth stage as a morpho-physiological indicator of Bromus valdivianus and Lolium perenne mixed pasture defoliation in New Zealand grazing system : 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, 2022) García Favre, Javier HoracioBromus valdivianus Phill. is a perennial grass species native to the South of Chile. Its better fitness for growing under low water availabilities compared with Lolium perenne L. has been assessed in field and glasshouse studies. However, Bromus valdivianus morpho-physiological attributes, such as root development under field conditions, and competitive ability are likely to manifest differently under contrasting defoliation criteria based on leaf regrowth stage. In addition, Lolium perenne and B. valdivianus growth can be complementary throughout the year under rainfed conditions. Therefore, the objective of the present thesis was to determine defoliation criteria based on leaf regrowth stage of mixed pastures (50/50% L. perenne and B. valdivianus) and consequences for species succession and functional traits development of the species. Four studies were developed in the thesis, two glasshouse and two field studies. The first glasshouse study evaluated the growth response and water-soluble carbohydrate (WSC) accumulation of B. valdivianus at three different defoliation frequencies. The results suggested that B. valdivianus increased herbage mass production and root biomass at low defoliation frequency (i.e., at 3.5–4.0 leaf regrowth stage). This was related to a higher WSC accumulation in the tiller base. In addition, under low soil water availabilities (20–25% of field capacity) WSC increased by ~20%, which indicated a drought resistant strategy of this species. The second study proved the growth enhancement (mainly root length and biomass) of B. valdivianus under competition with L. perenne compared with intraspecific competition when soil water shifted from high to low availabilities. In field studies, B. valdivianus mixed with L. perenne increased ~15% accumulated herbage mass compared to the species monocultures, which supported higher production during dry periods. This was due to the niche complementarity and asynchrony in herbage growth between the species, with B. valdivianus capable to maintain a steady tiller population throughout the year and root biomass accumulation at depth. Whereas L. perenne grew more than B. valdivianus under low level of oxygen in the soil and under optimal growth conditions and presented a higher nutritive value than B. valdivianus during winter, spring and autumn. The latter supports the partial grazing preference for L. perenne shown by sheep during part of the year, as measured in the second field study. This lower nutritive value (less energy) of B. valdivianus monocultures was overcame in the mixture, with a good overall value for high animal production. Regarding the mixture defoliation criterion, it was shown that it can be based on the optimal leaf regrowth stage of either species, as herbage mass production was similar between defoliation frequencies based on optimal leaf regrowth stage of either of the species. However, under defoliation based on optimal leaf regrowth stage of B. valdivianus, root biomass accumulation at depth increased ~45%. Overall, the results of the present thesis evidenced that Bromus valdivianus can successfully grow alongside L. perenne and, therefore, the mixture can increase forage production of New Zealand farming system in the event of climate change.Item Water-use efficiency in perennial ryegrass (Lolium perenne L.) : 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, 2021) Weerarathne, Lekamalage Visna YahaniKnowledge of genetic and physiological bases of drought responses and stress tolerance properties of pasture plants is an integral part of designing efficient pasture improvement programs to combat the consequences of climate change. However, experimental evidence or theoretical analyses on that aspect is sparse in the literature. Therefore, considering wider applications, high economic importance, and acknowledged poor tolerance of (Lolium perenne L.; PRG) to drought, the main aim of this research was to evaluate morpho-physiological trait responses that are linked to water-use efficiency (WUE) in different PRG populations from different sources in simulated summer drought cycles under a controlled environment. This study also included a quantitative genetic analysis conducted on the key traits to ascertain which traits are under genetic control for future breeding purposes. Experiment 1 screened single potted PRG genotypes from three commercial cultivars with industry reputation for persistence for natural differences in morpho-physiological traits related to water use (WU) under simulated drought. Large within-population variation was observed for the measured traits which included, among others, WUE (g WU/g plant dry matter); shoot dry weight, SDW; leaf osmotic potential, OP; leaf relative water content; predawn leaf water potential; root dry weight at 20–50 cm depth, RDWD; gravimetric soil moisture at 30–40 cm depth, SMCD; post-cutting regrowth. Principal component analysis (PCA) identified important trait associations contributing to high WUE (i.e. WUE-OP-RDWD trait association) and one related to higher SDW together with ‘SMCD conservation’ indicative of ‘true WUE’ was used to make a divergent selection of 20 high- and 15 low-WUE genotypes (HWUE and LWUE, respectively). Experiments 2 and 3 were conducted simultaneously using the same methodology as Experiment 1 and inter-randomised in the same glasshouse space. Experiment 2 retested clones of HWUE and LWUE plants selected in Experiment 1 for consistency of trait expression across the two consecutive growing seasons, and also collected data for additional traits. Results confirmed that the key trait associations identified in Experiment 1 were almost identically expressed in Experiment 2. From data on additional traits, it was established that the accumulation of high molecular weight sugars in the shoots significantly contribute to ‘true WUE’ of a subset of PRG genotypes but, with the less involvement of gas exchange data under the conditions tested. It is speculated that enhanced mesophyll conductance of CO2 might underlie this important trait association. However, the large majority of genotypes exhibited a ‘SMCD-depleting’ trait association of WUE with improved gas exchange and maximum quantum efficiency of PSII, demonstrating late A.R. Blum’s theory of WUE. Thus, selection of PRG for drought tolerance should consider yield and soil moisture data together to establish the most appropriate category of WUE trait association in improved cultivars. Experiment 3 investigated drought response trait associations in two further populations: a group of elite plants from a commercial plant breeder’s breeding program (CBL) and a PRG germplasm line derived from crossing Mediterranean and Middle Eastern seed accessions (MMEL) compared with those from the HWUE selection. PCA results showed that the major trait associations found in the yield and water relations data of elite subsets of CBL closely followed those of the HWUE selection, but elite MMEL plants exhibited typical summer dormancy characteristics where the average SDW of MMEL was 40% of lower than that of the CBL plants. Results further suggested that the company field evaluation system could benefit from the consideration of water relations traits, including WUE and associated traits like OP, as externally-measured selection traits for PRG drought tolerance. Experiment 4 evaluated quantitative genetic parameters of the key traits using the same methodology from Experiment 1 in a breeding population of 36 ‘half-sib (HS) families’ under both stressed and non-stressed conditions. There were significant estimates of among- and within-HS family genetic variances, narrow-sense heritability, and predicted genetic gain estimates for the key traits, indicating high genetic potential of each trait for breeding purposes under the conditions tested. However, the correlated response to selection of each trait pair comprising highly genetically correlated morpho-physiological traits with WUE was lower than that gain from the single-trait selection, accommodating further research questions on the efficacy of indirect and multi-trait selection of key traits. Based on the current results, it was found that the direct selection of PRG for WUE or concurrent selection for OP, RDWD, SMCD, and SDW or RGS traits under drought is advisable. Furthermore, significant quantitative genetic parameters estimated for WUE under non-stressed conditions together with high genetic correlation observed for WUE between stressed and non-stressed conditions suggested that a PRG population can potentially be selected for this trait at early growth stages or before imposition of water deficit.Item Genetic signatures in a perennial ryegrass (Lolium perenne) population following recurrent selection for compatibility with an endophyte (Epichloë spp.) from tall fescue : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Breeding at Massey University, Palmerston North, New Zealand(Massey University, 2019) Pocsedio, Arnel E.Perennial ryegrass or Lolium perenne L. (Poaceae) is the most extensively grown forage especially in the temperate regions of the world, including New Zealand. The development of forage cultivars is important to New Zealand since the livestock industry depends on perennial ryegrass for its nutrition needs. Among forage breeding objectives, persistence is particularly complex. It refers to the stability of dry matter yield over time. It is economically important because reseeding and cultivation can be capital-intensive. Persistence is partly modulated by the interaction of perennial ryegrass with Epichloë spp. as these fungal endophytes confer insect resistance for a more stable yield. Genetic factors in the host influence fungal biomass, alkaloid concentration, and endophyte vertical transmission frequency. The symbiotic relationship is therefore exploited in perennial ryegrass breeding. Thus, the objective of this study is to investigate a perennial ryegrass breeding population under recurrent selection (RS) for compatibility with an endophyte sourced from tall fescue. Specifically, this study aims to (1) investigate the transmission of the Epichloë sp. FaTG-3 strain AR501 in the breeding population PGG04, and (2) to examine how genetic variation changes during RS in terms of population differentiation. Since the selection program targets endophyte compatibility, signatures of selection that may be associated with the grass-endophyte interaction were also determined. It was hypothesized that: there will be a reduction of diversity, and an excess of rare alleles. Furthermore, it was hypothesized that loci under positive selection will have higher fixation index (FST), and their genotypes will be more correlated with the components of PCA- based population structure analysis compared to neutral loci. The presence of AR501 was examined in seeds, in the growing tillers, and by microsatellite genotyping for both the early and late generations of PGG04. The seed squash assay revealed that more than 90% of PGG04 seeds harboured the endophyte, regardless of the generation. Viable endophyte detection using tissue-print immunoblotting showed an increase in infection from ca. 5% to 33% between the early and late generations. Thus, the results suggest that positive selection for endophyte compatibility increased the proportion of viable endophyte in the population. This study provides evidence supporting host genetic control of the association in grass-endophyte interaction, and that this can be exploited in plant breeding programs. Changes in the genetic variation of PGG04 was investigated by comparing GBS data of the early and late generations. Results showed that selection enriched the late generation with rare alleles (0.02 - 0.08) compared with the early generation. Also, selection reduced expected heterozygosity from 0.3069 in PGG04-C2 to 0.3033 in PGG04-C6. Further, selection changed the population structure based on UPGMA dendrogram, PCA, and the model-based clustering method implemented in STRUCTURE. A few single nucleotide polymorphisms (SNPs) have relatively larger contribution to the population structure changes hence, they have relatively high FST, and their genotypes correlated with principal components. Logistic regression of these SNPs with infection data identified nine SNPs to be associated with the trait. Depending on the allele frequency, these SNPs can increase the odds of favourable infection by more than five times. Annotation of these SNPs identified S7_160751877 to be tagging an ABCG transporter gene. Since some ABC transporters mediate plant-microbe interactions, it is possible that the identified SNPs are tagging a gene involved in the host genetic control of grass-endophyte interaction.Item A study of the early development of the root systems of various grass species, and, a study of the importance of various roots, particularly the seminal roots to Lolium perenne : thesis submitted in part fulfilment for the degree of Master of Agricultural Science in Plant Husbandry, University of New Zealand(Massey University, 1950) Yates, Milton EarleNew Zealand is essentially a land of pasture. Greater reliance is placed on pastures for the sustenance of stock by the farmer in New Zealand than probably anywhere else in the World. The study of pastures is theretore of paramount importance to the national welfare of New Zealand and their improvement will be reflected in raised living standards and at the same will provide more food for the hungry world of today. There is no doubt that New Zealand is favoured by an equable climate and well distrtbuted rainfall which enables her to derive a considerable portion of her wealth trom grasslands. Nevertheless we have had to adopt farming methods to suit the environment so that the greatest benefits may be derived from the natural advantages with which New Zealand is endowed. New Zealand has proceeded a long way since the early days of her farming when little was known of the environmental requirements of individual species in order that they may produce to their maximum. It is unquestionable that much of the progress already made in grassland husbandry in this country is due to the wide range of trials conducted by the Department of Agriculture and Grasslands Division, D.S.and I.R., particularly over the last quarter of a century. Much has been learnt in the past and investigations being pursued at the present time will no doubt be reflected by improved methods of grassland husbandry with consequent increased pasture production in the future. [From Introduction]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 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 Exploiting heterosis in perennial ryegrass (Lolium perenne) through development of inbred lines, and the impact on population variability : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Agricultural Science, Massey University(Massey University, 2018) Brok, Louise NicoleGenetic improvements in the dry matter yield of perennial ryegrass via plant breeding are typically achieved through recurrent selection, delivering rates of genetic gain estimated to be in the range of 0.25 - 0.76% per year. Hybrid breeding is commonly used in self-compatible species (e.g. maize) to achieve significant yield increases through the exploitation of heterosis. However, hybrid breeding has not been used to a large extent in perennial ryegrass, due to its self-incompatibility (SI) system. However, using marker assisted selection (MAS), the alleles responsible for SI in perennial ryegrass can now be manipulated. A method has been developed which uses MAS to develop parent lines with controlled SI alleles, which are inbred for two cycles and are then crossed to create hybrids. This method provides the opportunity to exploit heterosis in perennial ryegrass breeding and for significant gains in dry matter yield. The first experiment in this thesis aimed to investigate the expression of heterosis in F1 hybrid plants produced by this proposed novel SI hybrid breeding method. It was expected that the hybrid offspring would at least display mid-parent heterosis. Experiment one also investigated the variability in key morphological traits, in the expectation that the cycles of inbreeding would have increased genetic uniformity in the parent lines and hybrids. The hybrids did display mid-parent heterosis throughout the experiment, providing evidence that the proposed method successfully captures heterosis in the perennial ryegrass breeding cycle. Evidence of high-parent heterosis were also observed throughout the experiment, which indicates the potential to develop F1 hybrids with significant yield increases compared to current cultivars. Therefore, the method may be commercially viable. No consistent changes in the morphological variation of the parent lines or hybrids was observed, which is a positive outcome for the ecology of perennial ryegrass in grazed pasture communities. The second experiment investigated expression of heterosis in F1 hybrid offspring from pairs crosses with different genetic backgrounds. The amount of variation in heterosis within each F1 hybrid population was also investigated. It was expected that expression of heterosis would vary dependent on the genetic background and that there would be significant variation in expression of heterosis within each F1 population. The expression of, and variation in, heterosis was of interest because with the advent of the SI hybrid breeding method, breeders may benefit from quantifying the combining ability of their perennial ryegrass breeding pools. This would enable better selection of plants for entry into the hybrid breeding pipeline. Mid-parent and high-parent heterosis were detected, but the levels of expression were variable within, and between, the two genetic backgrounds. This supports the hypothesis that there is variation in the performance of hybrids with differing genetic backgrounds, and therefore, there would be value in quantifying the combining ability of perennial ryegrass breeding pools.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.