Abstract
We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N5-trans-anhydromevalonyl-N5-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.
Highlights
Iron is an essential nutrient for almost all organisms due to its central role in vital cellular reactions
The axenic vegetative growth characteristics of the DsidN E. festucae mutants were examined on defined medium (DM) and on DM supplemented with the ferrous iron chelator bathophenanthrolinedisulfonic acid (BPS), which is impermeable to cell membranes [54,55] (Figure 1A)
The lack of growth by the mutants on this medium is explainable by the loss of both reductive iron assimilation (RIA)-mediated and siderophore mediated iron uptake from the extracellular medium, and demonstrates the presence of RIA in E. festucae
Summary
Iron is an essential nutrient for almost all organisms (except for some lactobacilli [1]) due to its central role in vital cellular reactions. The redox properties of iron confer a catalytic function essential for fundamental metabolic pathways such as DNA synthesis, respiration and photosynthesis [2,3]. Even though iron is a highly abundant metal, in aerobic environments bioavailability is low because ferric iron forms insoluble oxides through reaction with oxygen. Controlling iron homeostasis is an essential function and organisms have developed complex strategies for iron uptake, utilization, transport and storage [5]. The uptake of iron is presumably the principal regulatory point of iron homeostasis and multiple high- and low-affinity iron uptake systems have evolved [6]
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