cAMP Signaling Regulates Synchronised Growth of Symbiotic Epichloë Fungi with the Host Grass Lolium perenne

Abstract

The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling.

Keywords: Epichloë festucae; Lolium perenne; cAMP signaling; hyphal branching; reactive oxygen species; symbiosis.

Figure 1

Disruption of acyA in E. festucae Fl1. (A) Domain architecture of AcyA in relation to the insertion site of the hygromycin resistance cassette (HygR). The protein contains the domains for G-alpha binding (G), ras association (RA), leucine rich repeats (L), protein phosphatase 2C (PP2C), and catalytic cyclic nucleotide biosynthesis (CYC) (Baker and Kelly, 2004). The HygR cassette was inserted (arrow) upstream of the phosphatase 2C (PPC2) and catalytic (CyC) domains. (B) Diagram of the acyA disruption locus in E. festucae Fl1. In vector pCRVacyhph, the HygR cassette is flanked by approximately 3 kb of DNA from the partial acyA gene (hatched) cloned from E. festucae var. lolii Lp19 (KR815911). The stop codon in the hygromycin resistance gene is indicated (⋆). The regions of homology between the Lp19 flanking regions and the corresponding Fl1 acyA sequence (hatched) is shown immediately above the Fl1 acyA gene, gene model Fl1M3.048730 (http://www.endophyte.uky.edu). Probes 1 and 2 (solid bars) were used in Southern-blot hybridization experiments (C) to confirm that the endogenous acyA gene had been disrupted and to ascertain the number of acyA copies in each strain respectively. (C) Southern-blot hybridization to confirm acyA insertion mutants. Genomic DNA was isolated from the wild-type (WT) plus a number of putative insertional mutants, and digested with HindIII. Probe 1 bound to the predicted 4.7 kb fragment in the wild-type acyA also in ectopic integrants acyA19 and acyA49 and to the 1.3 kb HindIII fragment of the disrupted acyA gene in ectopic integrants acyA19 & acyA49 and insertional mutants ΔacyA34 (Δ34), ΔacyA42 (Δ42) and ΔacyA47 (Δ47). Probe 2 was used to determine copy number, and bound to either a 4.4 kb or 4.5 kb HindIII fragment depending on whether the recombination locus was before or after the 66 bp indel in the Lp19 acyA.

Figure 2

Growth of E. festucae acyA disruption mutants in axenic culture. (A) Growth of wild-type, disruption mutants ΔacyA34 (Δ34), ΔacyA42 (Δ42), ΔacyA47 (Δ47), and colonies with an intact acyA gene plus an ectopic insertion(s) of the transformation vector, acyA19, acyA49 on PDA (22°C for 7 days). (B) The effect of exogenous cAMP on the radial growth rate of the same strains. The mean growth rate of each strain (with three clonal replicates) at each cAMP concentration is shown. Analysis of variance was used to compare the growth rate of strains between and within each concentration of cAMP. The vertical bar represents the least significant differences (LSD) between means at the 5% significance level when comparing strains at the same concentration of cAMP.

Figure 3

Effects of acyA disruption on E. festucae hyphal morphology in axenic culture. Bright field images of E. festucae Fl1 hyphae growing on water agar at 22°C for 7 days (bar, 10 μM). Images were taken approximately 3 mm behind the colony margin. Hyphae of all independent mutants (ΔacyA34, ΔacyA42, and ΔacyA47) were convoluted, irregular in diameter (ΔacyA47) and heavily branched compared to the wild-type. Ectopic integration of the wild-type acyA gene into ΔacyA34 and ΔacyA42 resulted in a reversion to the wild-type phenotype to greater or lesser extents dependent on the individual strain (ΔacyA34/acyA, ΔacyA42/acyA). Gross morphology at the hyphal tips appeared similar in mutants and wild-type.

Figure 4

Influence of acyA disruption on production of ROS in E. festucae Fl1 hyphae in axenic culture. Bright field images of E. festucae Fl1 growing on PDA at 22°C for 7 days and stained with NBT. Included is a low (scale bar = 50 μm) and high (scale bar = 5 μm) resolution image of the wild-type (A) plus mutants ΔacyA34 (D), Δ42 (E), and Δ47 (F), and complementation strains ΔacyA34/acyA (B) and ΔacyA42/acyA (C).

Figure 5

Morphology of E. festucae ΔacyA in L. perenne. Aniline blue-stained hyphae (arrows) in the mature second leaf sheath of L. perenne infected with the wild-type and strains ΔacyA34 and ΔacyA42. The morphology of the mutant strains after complementation (ΔacyA34/acyA and ΔacyA42/acyA) with the wild-type gene is also shown. All images are at the same magnification. The scale bar is 5 μm.

Figure 6

Disruption of E. festucae acyA deregulates hyphal branching during host colonization. (A–J). Confocal laser scanning micrographs (CLSM) of E. festucae hyphae at three stages of host development. L. perenne leaves infected with the wild-type and disruption mutant ΔacyA42 are shown. Both strains are transformed with plasmid pTEFEGFP and constitutively express EGFP. (A′–J′), CSLM overlaid on corresponding phase contrast images of the same field of view. (K) L. perenne tiller showing the positions at which the tissue sections were taken. A/B, CLSM of a longitudinal section of the mature upper leaf sheath of the pseudostem 3.5 cm above the crown (base) of the tiller showing colonization by WT and ΔacyA42 hyphae respectively. (C,D) CLSM of a transverse section though the tiller at the same position as (A,B). The youngest developing leaf is in the center of the section and is enclosed by progressively older leaf sheaths. (E,F) CLSM of a longitudinal section of a young developing leaf sheath immediately above the crown of the tiller. (G,H) CLSM of a transverse section through the pseudostem at the same position as (E,F). (I,J) CSLM of a longitudinal section through the crown showing the position of hyphae relative to the shoot apex. The shoot apex (red arrow) is a small dome of overlapping leaf primordia visible only in the phase contract images (I′, J′). Hyphae are either not detectable in this tissue by confocal microscopy or are not present. Hyphae in the youngest leaves emerging from the shoot apex are marked by white arrows.

Figure 7

Effects of acyA disruption on biomass of E. festucae in host grasses. Comparison of hyphal biomass in the pseudostem (the region between the crown and the first ligule of a tiller) of plants infected with either the wild-type or ΔacyA42 mutant strains. Hyphal biomass (expressed as endophyte concentration) was determined by quantitative PCR of a single copy gene from 1 ng of genomic DNA. Hyphal biomass between strains was compared using the Students' T-test. The means of the two treatments were significantly different (P = 0.004).

Figure 8

Regulation of hyphal cell wall synthesis by cAMP during leaf colonization. The cell wall of 12 hyphae each from developing leaves (A) and mature second leaf sheaths (B) of L. perenne was measured at eight positions around each hypha. The gray symbols represent the average cell wall thickness of each hypha. The overall mean cell wall thickness and its 95% confidence interval is presented for each strain. Analysis of variance was used to compare the strains. Within each plant tissue (A,B), different letters indicate means that are different at the 5% significance level. Transmission electron micrographs show representative hyphae of E. festucae wild-type, mutants ΔacyA34, and ΔacyA42, and the complementation strain ΔacyA34/acyA strain in the two host tissue types. All images are at the same magnification. The scale bar is 500 nm.