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

Abstract

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 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.