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Item Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne(The American Phytopathological Society in cooperation with the International Society for Molecular Plant-Microbe Interactions, 2021-10-28) Noorifar N; Savoian MS; Ram A; Lukito Y; Hassing B; Weikert TW; Moerschbacher BM; Scott BEpichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.Item A conserved signalling network regulates Epichloë festucae cell-cell fusion and the mutualistic symbiotic interaction between E. festucae and Lolium perenne : 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, 2016) Green, Kimberly AnneEpichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. The NADPH oxidase complex components noxA, noxR and racA, the transcription factor proA, and the cell wall integrity (CWI) MAP kinases, mkkA and mpkA, are required for mutualistic E. festucae-L. perenne associations and cell-cell fusion. Homologues of these genes in Neurospora crassa, Sordaria macrospora and Podospora anserina are required for cell-cell fusion and sexual fruiting body maturation, thereby establishing a link between self signalling and hyphal network formation in the E. festucae-L. perenne symbiosis. In Podospora anserina, IDC2 and IDC3 are required for cell-cell fusion, crippled growth and fruiting body formation. In S. macrospora and N. crassa, components of the STRIPAK complex regulate cell-cell fusion and fruiting body formation. The aim of this project was to test if E. festucae homologues of IDC2 and IDC3, and the STRIPAK complex protein MOB3, named SymB, SymC and MobC, respectively, are also required for cell-cell fusion and plant symbiosis. Gel shift assays showed the promoters of symB and symC are targets for the transcription factor ProA. In culture, the frequency of cell-cell fusion of ΔmobC was reduced, but in ΔsymB and ΔsymC mutants, totally abolished. All three mutants hyperconidiated and formed intra-hyphal hyphae. Plants infected with these mutants were severely stunted and hyphae exhibited proliferative growth and increased colonisation of the intercellular spaces and vascular bundles. Expressoria formation, structures allowing colonisation of the leaf surface, was reduced in ΔmobC, and abolished in ΔsymB and ΔsymC mutants. Microscopy analyses showed SymB-GFP and SymC-mRFP1 co-localise to the plasma membrane and septa. SymC also localised to highly dynamic punctate structures. Although ΔsymB and ΔsymC phenotypes are identical to ΔmpkA, and the E. festucae pheromone response pathway scaffold ΔidcA mutants, MpkA and MpkB phosphorylation and cellular localisation was unchanged compared to wild-type. Using yeast-two-hybrid assays, an interaction between SymC and the STRIPAK complex associated protein GPI1 was demonstrated. Collectively these results show that MobC, SymB and SymC are required for E. festucae cell-cell fusion and host symbiosis. It is proposed that SymB and SymC interact to form a sensor complex at the cell wall which regulates cell-cell fusion in culture and hyphal network development in planta.Item Characterisation of novel secondary metabolism genes in plant-endophytic Epichloë fungi : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand(Massey University, 2016) Berry, DanielPlant-‐endophytic fungi of the genus Epichloë are symbionts of cool season grasses. Epichloë infections are generally asymptomatic, with endophyte and host growth synchronised to form an interconnected hyphal network in the intercellular spaces separating host cells. In return for the sustenance received from the host apoplast, Epichloë spp. produce a range of bioprotective secondary metabolites (SM) that deter both vertebrate and invertebrate herbivores. Peramine is an Epichloë-‐ derived insect-‐deterring SM produced by the two-‐module non-‐ribosomal peptide synthetase (NRPS) PerA, encoded by the gene perA. The perA gene is widespread across Epichloë, but peramine production is much more discontinuous. Through an Epichloë-‐wide survey it is shown that the peramine chemotype of Epichloë isolates can be accurately predicted through a combination of PCR and sequencing methods. Furthermore, the protein encoded by the widespread peramine-‐negative perA-‐ΔR allele is analysed in detail. The perA-‐ΔR allele has a transposable element inserted into the 3’ end of the gene, and was previously assumed to be non-‐functional. Evidence for ongoing perA-‐ΔR functionality is presented, with the PerA-‐ΔR protein shown to exhibit altered substrate specificity relative to PerA. A PerA/PerA-‐ΔR domain swap is shown to restore peramine production to PerA-‐ΔR, providing insight into the functional changes that separate this protein from PerA. Another major project investigated the role SM genes may play in Epichloë sexual development. Many Epichloë spp. are transmitted vertically through infection of the host seed. However, sexual Epichloë spp. may also undergo a reproductive cycle that involves formation of pre-‐sexual stromata structures over developing host inflorescences. This sexual cycle culminates in horizontal transfer of the endophyte to endophyte-‐free host plants. Deletion of the symbiotically regulated endophyte gene irlA induced a symbiosis-‐defective phenotype in planta, and this observation led to the identification of a novel Epichloë SM cluster. Synteny analysis and comparison to previously characterised fungal SM gene clusters defines a five-‐gene cluster centred on irlA that is shown to be upregulated in stromata. A model is proposed whereby this SM cluster controls initiation of the proliferative hyphal growth associated with stromata formation.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.