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
Loading...
Date
2019
DOI
Open Access Location
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
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.
Description
The following Figures were removed for copyright reasons: 1.5 p.32 (=Christensen et al., 2008 Fig 3c d h), 1.5 p.35 (=Christensen et al., 2008 Fig 2a b c d e) & 1.8 p.40 (=Kock et al., 2015 Fig 2). Other copyrighted Figures remain for clarity's sake.
Keywords
Epichloë, Growth, Genetics, Effect of stress on, Calcium, Physiological effect, Fungal cell walls