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
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2019
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Massey University
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Abstract
Fungi 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.
Description
Listed in 2019 Dean's List of Exceptional Theses
Figures re-used with publishers' permission.
Figures re-used with publishers' permission.
Keywords
Epichloë, Lolium perenne, Genetics, Endosymbiosis, Plant-fungus relationships, Plant cellular signal transduction, Dean's List of Exceptional Theses