Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction

Abstract

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae-perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction.

Figure 1.

Symbiotic and Axenic Culture Phenotypes of E. festucae FR2. (A) Phenotypes of perennial ryegrass infected with E. festucae wild-type Fl1, symbiotic mutant FR2, and complemented strain C8. The photograph was taken 9 weeks after inoculation. (B) Colony morphology of E. festucae wild-type Fl1 and symbiotic mutant FR2 on complete medium agar after 10 d. (C) Confocal depth series image of hyphal growth of wild-type WG11 expressing GFP in the leaf sheath of perennial ryegrass. The photograph was taken 10 weeks after inoculation. Bar = 20 μm. (D) Confocal depth series image of hyphal growth of symbiotic mutant FR2G6 expressing GFP in the leaf sheath of perennial ryegrass. The photograph was taken 10 weeks after inoculation. Bar = 20 μm.

Figure 2.

In Planta Phenotype of E. festucae Symbiotic Mutant. (A) to (D) Light micrographs of transverse sections of the outer leaf of perennial ryegrass stained with toluidine blue. Hyphae of endophyte are indicated by arrowheads. Insets in (B) to (D) show higher magnification images of the endophyte hyphae indicated by open arrowheads in the main panels. Bars = 20 μm. (A) Endophyte-free leaf of perennial ryegrass. (B) Leaf infected with wild-type Fl1. (C) Leaf infected with symbiotic mutant FR2. (D) Leaf infected with complemented strain C8. (E) and (F) Transmission electron micrographs of cross sections of endophyte hyphae in the intercellular space of the host plant. c, crystalline aggregation; fcw, fungal cell wall; m, mitochondrion; v, vacuole. Bars = 0.25 μm. (E) Wild type. (F) FR2 mutant.

Figure 3.

Physical Maps of Plasmid-Tagged Mutant and Wild-Type Loci. The pAN7-1–tagged locus in the FR2 mutant (pPN72) and the corresponding wild-type genomic region (pPN70) showing the plasmid insertion site in the mutant and restriction enzyme sites for BglII (B), ClaI (C), EcoRI (E), EcoRV (V), HindIII (H), SalI (S), XbaI (Xb), and XhoI (Xh). The arrow identifies the E. festucae noxA gene. Sequence analysis of the junctions between pAN7-1 and E. festucae noxA in FR2 showed that the HindIII site at the right junction of the plasmid and genomic DNA was lost.

Figure 4.

Alignment of the Predicted Amino Acid Sequences for E. festucae noxA and noxB with Related Proteins. The amino acid sequences of E. festucae (Ef) NoxA and NoxB are aligned with those of human (Hs) gp91^phox (Royer-Pokora et al., 1986), A. nidulans (An) NoxA (Lara-Ortíz et al., 2003), and P. anserina (Pa) Nox1 and Nox2 (Lalucque and Silar, 2003). Amino acids conserved between sequences are boxed in black (identical) or gray (conservative replacements). Six potential transmembrane-spanning domains (TM1 to TM6) and putative FAD and NADPH binding domains are indicated by lines above the aligned sequences. Conserved His residues, demonstrated to bind heme in yeast ferric reductase and human gp91^phox (Finegold et al., 1996), are indicated by asterisks. Dots under the sequences indicate amino acid residues involved in the N-glycosylation of human gp91^phox (Wallach and Segal, 1997).

Figure 5.

Deletion of E. festucae noxA and noxB. (A) Physical map of the E. festucae noxA and noxB genomic regions, pPN70 and pPN71, and linear inserts of replacement constructs, pPN75 and pPN78, showing restriction enzyme sites for ApaI (A), BglII (B), ClaI (C), EcoRI (E), EcoRV (V), HindIII (H), SalI (S), XbaI (Xb), and XhoI (Xh). (B) Autoradiographs of a DNA gel blot of SacII genomic digests (2 μg) of E. festucae wild-type strain Fl1 (WT) and noxA deletion strains A17 and A44 probed with digoxigenin-labeled noxA insert from pPN74 amplified with primers pII99-3 and pII99-4, and of a DNA gel blot of ApaI genomic digests of E. festucae wild-type strain Fl1 (WT), noxB deletion strain B7, noxA deletion strain A44, and noxA noxB double deletion strain A44.B29 probed with digoxigenin-labeled noxB insert from pPN71 amplified with primers nox2a and nox2d. Sizes in kilobases of HindIII-digested λDNA markers are indicated at left. (C) and (D) Phenotypes of perennial ryegrass plants infected with E. festucae wild-type Fl1 and noxA deletion strain A44 (C) and noxB deletion strain B7 and noxA noxB double deletion strain A44.B29 (D). The photographs were taken 9 weeks after inoculation.

Figure 6.

Expression Analysis of Endophyte and Plant Genes. (A) Expression analysis of E. festucae nox genes. Total RNA was isolated from pseudostems of Fl1-infected perennial ryegrass (in planta) or from mycelia of Fl1 grown in axenic culture on potato dextrose medium (in culture) and used for cDNA synthesis. RT-PCR was performed with primers specific for E. festucae noxA (Ef noxA), noxB (Ef noxB), and β-tubulin (Ef tubB). To compensate for the differences in endophyte biomass in planta versus that in culture, a 1:80 dilution of the Fl1 cDNA was used compared with undiluted pseudostem template. (B) Expression analysis of L. perenne PR genes in host plant infected with Fl1 or the noxA mutant. Total RNA was isolated from emerging leaf of Fl1-infected wild-type (WT) or noxA mutant–infected (A44) perennial ryegrass. Expression of L. perenne actin (Lp Act1), PR1 (Lp PR1), and PR5 (Lp PR5) and E. festucae noxA (Ef noxA) and β-tubulin (Ef tubB) were analyzed by RT-PCR with specific primers.

Figure 7.

ROS Production by E. festucae Wild Type and nox Mutants. (A) Detection of superoxide production by NBT staining. Photographs of colony mycelia of E. festucae wild-type Fl1 (WT), noxA mutant (A44), noxB mutant (B7), and noxA noxB double mutant (A44.B29) grown on potato dextrose agar medium and stained with 0.05% (w/v) NBT solution for 5 h. Bars = 2 mm. (B) and (C) Higher magnification light micrographs of hyphae in (A) showing localization of reduced NBT at the hyphal tip (B) and in a subapical cell at the end proximal to the growing tip (C). The direction of the hyphal growing tip in (C) is indicated by the arrow. Bars = 10 μm. (D) to (H) Transmission electron micrographs of H₂O₂ localization in meristematic tissue of perennial ryegrass infected with E. festucae wild-type Fl1 (D) to (F) or noxA mutant A44 (G) and (H). Cerium chloride–reactive deposits are indicated by arrowheads. ecm, extracellular matrix; pc, plant cell. Bars = 1 μm. (I) Distribution of five different types of cerium chloride–reactive deposits showing H₂O₂ localization in meristematic tissue of perennial ryegrass infected with E. festucae wild-type Fl1 (WT) or noxA mutant (A44). Type 1, cerium chloride–reactive deposits in the extracellular matrix of endophyte; type 2, cerium chloride–reactive deposits in host cell walls; type 3, cerium chloride–reactive deposits in both endophyte extracellular matrix and host cell walls; type 4, cerium chloride–reactive deposits in plasma membrane of endophyte; type 5, no deposit detected. The number of fungal cells of each particular type is given above each column.