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

Epichloë 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 intracellular 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 conducted 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.