Role of iron, siderophores and iron regulation in the Epichloë festucae - Lolium perenne symbiosis

Ferric iron-chelating siderophores are produced by microorganisms to compete for and sequester iron, an essential but potentially toxic micronutrient. Epichloё festucae, a filamentous fungus that is mutualistically associated with the grass Lolium perenne, occupies apoplastic spaces between the plant’s shoot cells and relies upon host nutrients to survive. E. festucae 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 E. festucae with L. perenne. Little else is known about the mechanisms governing iron metabolism in E. festucae or the role of iron in maintaining E. festucae - grass associations. To explore siderophore biosynthesis and iron regulation in E. festucae, iron-related genes were identified and fungal mutants were generated. These mutants, deficient in either siderophore biosynthesis (ΔsidA, ΔsidC, and ΔsidN) or iron regulation (ΔsreA and ΔhapX) were characterised and their phenotypes analysed in culture and in planta. The sidA gene encodes the L-ornithine-N5-oxygenase SidA, an enzyme shown to be required for the biosynthesis of both siderophores. The sidC 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 ΔsidN 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 ΔsidN compared to WT cultures, suggesting an increased intracellular iron pool in the ΔsidN 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 recompen se for the loss of FC, and repressed ΔsidN colony growth in excess, suggesting that epichloënin A modulates iron accessibility intracellularly. The ΔsidN 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 in planta fungal growth, which might prevent over-colonisation of important host organs such as meristems whose development affect plant architecture. The sreA and hapX genes encode two iron-responsive transcription factors that are involved in regulating iron homeostasis in E. festucae 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 L. perenne were not stably maintained long term and hyphae displayed atypical morphology. The ΔsreA fungi could induce chlorosis during low iron supply to host plants indicating that ΔsreA mutants compete with the host for iron. E. festucae appears to have a tightly regulated iron management system for b alancing growth and survival, preventing over-competition for iron in the intercellular niche thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively impact iron-dependent fungal growth and can destabilise mutualistic plant - fungal associations to the detriment of either symbiotic partner.