An Extracellular Siderophore Is Required to Maintain the Mutualistic Interaction of <i>Epichloë festucae</i> with <i>Lolium perenne</i>

<div><p>We have identified from the mutualistic grass endophyte <i>Epichloë festucae</i> a non-ribosomal peptide synthetase gene (<i>sidN</i>) 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 <i>sidN</i> eliminated biosynthesis of epichloënin A <i>in vitro</i> and <i>in planta</i>. During iron-depleted axenic growth, Δ<i>sidN</i> mutants accumulated the pathway intermediate N⁵-<i>trans</i>-anhydromevalonyl-N⁵-hydroxyornithine (<i>trans</i>-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of <i>Lolium perenne</i> (perennial ryegrass) with Δ<i>sidN</i> 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 Δ<i>sidN</i> 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.</p></div

Synthesis of epichloënin A is dependent on <i>sidN</i>.

<p>LC-MS analysis showing MS¹extracted ion chromatograms for both epichloënin A (a, <i>m/z</i> 542) and ferriepichloënin A (b, <i>m/z</i> 569) in supernatant and mycelium from two week old iron-depleted cultures of wild-type <i>E. festucae</i> Fl1 (WT), <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>), and a complemented <i>ΔsidN</i> strain (C-<i>sidN</i>). Note scale for supernatant is 10× of that for mycelium.</p

The Endophyte-Grass symbiotic interaction phenotype is disrupted in <i>ΔsidN</i> infected plants.

<p>A. Phenotypes of perennial ryegrass plants infected with wild-type <i>E. festucae</i> Fl1 (WT), complemented <i>ΔsidN</i> strain (C-<i>sidN</i>) and <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>). B. Root systems of <i>sidN</i> infected plants are reduced compared to WT infections. A perennial ryegrass plant infected with <i>E. festucae</i> (WT) is compared against three Δ<i>sidN</i> 85 infected plants displaying increased levels of plant stunting from left to right. Plants are 14 weeks old. C, D and E. Light micrograph DIC images of aniline-blue stained hyphae of wild-type <i>E. festucae</i> Fl1 (WT). C. <i>ΔsidN</i> mutant 85. D. <i>ΔsidN</i> mutant 54. E. in mature leaf sheaths.</p

Iron depletion renders Δ<i>sidN</i> mutants incapable of axenic vegetative growth.

<p>A. 16-day-old cultures of wild-type <i>E. festucae</i> Fl1 (WT) and Δ<i>sidN</i> mutant strains (<i>ΔsidN</i> 54 and <i>ΔsidN</i> 85) were grown on iron depleted defined media (DM), and DM media supplemented with 100 µM BPS, or 100 µM BPS and culture filtrate from WT (BPS CF), or 100 µM BPS and 20 µM FeS0₄ (BPS Fe²⁺) or 100 µM BPS and 20 µM FeCl₃ (BPS Fe³⁺) respectively. B. Radial growth measurements of WT, complement (C-sidN) and Δ<i>sidN</i> 85 were determined by inoculating mycelial plugs in triplicate onto DM media, or DM supplemented with 100 µM BPS, DM with 100 µM BPS and 20 µM FeS0₄ (Fe2+) and DM with 100 µM BPS and 20 µM FeCl₃ (Fe3+) respectively. Colonies were measured at 10 days. The results represent the mean of three independent experiments. The radial growth is normalized to that of WT grown on DM media.</p

RT-qPCR of <i>E. festucae</i> iron-regulated genes and <i>Nox</i> genes in <i>ΔsidN</i> infected perennial ryegrass.

<p>Relative mean abundance relative to wild-type (fold difference displayed) of iron regulated gene expression (<i>ftrA</i>, <i>fetC</i>, <i>hapX</i>) and Nox gene expression (<i>noxA</i>, <i>noxB</i>, <i>noxR</i>, <i>racA</i>) in perennial ryegrass infected with Δ<i>sidN</i> mutants 54 and 85 are shown. For <i>ftrA</i>, <i>fetC</i>, <i>noxA</i> and <i>racA</i> results have a p-value of <0.001 and for <i>hapX</i>, <i>noxB</i> and <i>noxR</i>, the p-values are 0.002, 0.005 and 0.017 respectively. Error bars indicate SED.</p

Abnormalities in the hyphal distribution and ultrastructure of <i>ΔsidN</i> mutants in perennial ryegrass plants.

<p>A. Light micrographs of 1 µM cross sections of the inner leaf sheath of perennial ryegrass infected with wild-type <i>E. festucae</i> Fl1 (WT) and <i>ΔsidN</i> mutant 85 (85) are shown. The top panel is a cross section of mesophyll cells, whereas the lower panel is a close up of vascular tissue. Representative hyphae are indicated by arrows and the circle on the 85 panel indicates epiphyllous hyphae. Inserts show higher magnification of the endophyte hyphae indicated by the arrowheads in the main panels. Bars = 20 µM. B. Transmission electron micrographs of cross sections of endophyte hyphae in the intercellular spaces of perennial ryegrass. Wild-type <i>E. festucae</i> Fl1 (WT), complemented <i>ΔsidN</i> strain (C-<i>sidN</i>) <i>ΔsidN</i> mutant 54 (54), <i>ΔsidN</i> mutant 85 (85) are shown. Samples shown were photographed from leaf sheath sections, with WT, C-<i>sidN</i>, and <i>ΔsidN</i> 85 hyphae located in mesophyll tissue, whereas <i>ΔsidN</i> 54 is in sclerenchma tissue. c, cytoplasm, v, vacuole. Bars = 500 nm.</p

<i>ΔsidN</i> mutants display abnormal hyphal morphologies on water-agar.

<p>A. Mycelia of wild-type <i>E. festucae</i> Fl1 (WT) on water agar. B. Mycelia of <i>ΔsidN</i> 85 mutant proliferating on water agar by lateral branches. C. Close up of <i>ΔsidN</i> 85 mutant mycelia showing hyphal convolutions and swellings. Bar is 50 µM.</p

Elevated ergovaline levels detected in Δ<i>sidN</i> infected plants.

<p>HPLC analysis of ergovaline production was carried out on perennial ryegrass pseudostems infected with wild-type <i>E. festucae</i> Fl1 (WT), <i>ΔsidN</i> mutant 85 (<i>ΔsidN</i>), and complemented <i>ΔsidN</i> strains (C-<i>sidN</i> 1 and C<i>-sidN</i> 2). The numbers of plant reps used for analysis were 3–5 for each sample. Error bars indicate standard deviation.</p

Colonies of <i>ΔsidN</i> mutants are sensitive to hydrogen peroxide on DM.

<p>Values given are ratios of radial growth measurements of colonies grown for 7 days at 22°C on DM (defined medium) or PD (potato dextrose) medium supplemented with 0.7 mM H₂O₂ versus DM or PD. Statistics were generated from an analysis of variance. LSD is the Least Significant Difference between any two means and the SED represents the Standard Error of the Difference.</p

Ultrastructural features of the Δ<i>sidN</i> mutant - plant infection phenotype.

<p><b>A.</b> Aberrant Δ<i>sidN 85</i> mutant hyphae in the mesophyll of the inner leaf sheath surrounding host cells. B. An epiphyllous Δ<i>sidN 85</i> mutant hypha on the outside of the outer leaf blade. C. Δ<i>sidN 85</i> mutant hyphae in the main vascular bundle of the inner leaf sheath. Bar indicates 1000 nm. fc indicates fungal cell, pc for plant cell, pcw for plant cell wall.</p