Role of iron, siderophores and iron regulation in the Epichloё festucae - Lolium perenne symbiosis
Forester, Natasha Talei
Ferric iron-chelating siderophores are produced by microorganisms to compete for and sequester iron, an essential but potentially toxic micronutrient. <i>Epichloё festucae</i>, a filamentous fungus that is mutualistically associated with the grass <i>Lolium perenne</i>, occupies apoplastic spaces between the plant’s shoot cells and relies upon host nutrients to survive. <i>E. festucae</i> synthesises two siderophores, epichloënin A and ferricrocin (FC). A non-ribosomal peptide synthetase SidN was previously found to be required for the biosynthesis of epichloënin A, for iron uptake, for normal colony growth under iron-starved conditions and for normal mutualistic associations of <i>E. festucae</i> with <i>L. perenne</i>. Little else is known about the mechanisms governing iron metabolism in <i>E. festucae</i> or the role of iron in maintaining <i>E. festucae</i> - grass associations. To explore siderophore biosynthesis and iron regulation in <i>E. festucae</i>, iron-related genes were identified and fungal mutants were generated. These mutants, deficient in either siderophore biosynthesis (<i>ΔsidA</i>, <i>ΔsidC</i>, and <i>ΔsidN</i>) or iron regulation (<i>ΔsreA </i>and <i>ΔhapX</i>) were characterised and their phenotypes analysed in culture and <i>in planta</i>. The <i>sidA</i> gene encodes the L-ornithine-<i>N</i>5-oxygenase SidA, an enzyme shown to be required for the biosynthesis of both siderophores. The <i>sidC</i> gene encodes an NRPS enzyme, SidC that assembles FC, a siderophore that was only found in mycelial fractions. Mass spectrometry data indicated that epichloënin A and FC are synthesised under low and high iron availabilities, respectively. Intriguingly, the findings of significant quantities of epichloënin A in mycelial fractions of the wild-type strain Fl1 (WT), combined with the observation that <i>ΔsidN</i> colonies lacked iron-dependent pigmentation suggested that epichloënin A functions in intracellular iron management, in addition to extracellular iron uptake. In the presence of iron, increased ferricrocin production and vacuolar-iron uptake occurred in <i>ΔsidN</i> compared to WT cultures, suggesting an increased intracellular iron pool in the <i>ΔsidN</i> colonies that might fuel the observed increase in aerial hyphal growth. Iron starvation impaired growth of all siderophore mutants in culture, but could be repaired by supplementing iron-deprived media with FC, which was shown to be required for aerial hyphal growth. Iron-bound ferriepichloënin A could not recompense for the loss of FC, and repressed <i>ΔsidN</i> colony growth in excess, suggesting that epichloënin A modulates iron accessibility intracellularly. The <i>ΔsidN</i> fungi grew profusely in hosts around meristematic tissues and correlated with a host-stunting phenotype, which could only be induced by loss of SidN (and epichloënin). These results suggest interplay between siderophores in moderating <i>in planta</i> fungal growth, which might prevent over-colonisation of important host organs such as meristems whose development affect plant architecture. The <i>sreA</i> and <i>hapX</i> genes encode two iron-responsive transcription factors that are involved in regulating iron homeostasis in <i>E. festucae</i> under iron-sufficient and -deficient conditions, respectively. Gene expression analyses showed an iron-dependent mutual control mechanism exists between SreA and HapX, and an alternative splicing mechanism may control SreA activity. Loss of SreA resulted in growth defects and de-repression of siderophore biosynthesis in the presence of iron, while loss of HapX raised FC levels during iron deficiency indicating repressor functions of these proteins. Associations of regulatory mutants with <i>L. perenne</i> were not stably maintained long term and hyphae displayed atypical morphology. The <i>ΔsreA</i> fungi could induce chlorosis during low iron supply to host plants indicating that <i>ΔsreA</i> mutants compete with the host for iron. <i>E. festucae</i> appears to have a tightly regulated iron management system for balancing growth and survival, preventing over-competition for iron in the intercellular niche thus promoting mutualistic associations. Mutations that interfere with <i>Epichloë</i> iron management negatively impact iron-dependent fungal growth and can destabilise mutualistic plant - fungal associations to the detriment of either symbiotic partner.
Advisor: Lamont, Iain L.; Johnson, Linda J.
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: siderophore; iron; Epichloё; symbiosis; mutualism
Research Type: Thesis