Fig 1.
A model showing the growth of endophytic fungus Epichloë festucae in host grass plants.
(A) Hyphal growth of GFP-labeled E. festucae strain Fl1 in meristematic tissue of perennial ryegrass. LP, leaf primordium. Bar = 100 μm. (B) Hyphal growth of GFP-labeled E. festucae strain Fl1 in pseudostem of perennial ryegrass. Points of hyphal fusion are indicated by arrowheads. Bar = 50 μm. (C) Growth zones of a grass leaf and growth pattern of endophyte hyphae. Hyphae of endophyte in host plant is shown as light blue lines. In division zone of grass plant, endophyte hyphae grow by tip growth, whereas middle part of endophyte hyphae extend and divide (intercalary extension) in elongation zone of host plant. Formation of lateral hyphal fusions are often observed (as shown in B). Adapted from Kavanová et al. [66] and Christensen et al. [11].
Fig 2.
Specific interactions of Epichloë festucae Rho GTPases, RacA and Cdc42, with components of Nox complex.
(A) Alignment of the deduced amino acid sequences for E. festucae Rho GTPases. Conserved domains among Rho GTPases are boxed. Amino acid substitutions introduced for constitutive active (CA) and negative (CN) form of small GTPases are indicated by arrows. (B) Phylogenetic Analysis of Rho GTPase from E. festucae. The tree was prepared by the neighbor-joining method (Saitou and Nei, 1987). The scale bar corresponds to 10 estimated amino acid substitutions per site. Numbers at the nodes indicate the percentage of 1000 bootstrap replicates that supported each labeled interior branch. Ef; Epichloë festucae, Fg; Fusarium graminearum, Mo; Magnaporthe oryzae, Nc; Neurospora crassa. (C) Yeast two-hybrid assays of the interactions between E. festucae NoxR, BemA and Rho GTPases. Rho GTPases have mutation in C-terminal plasma membrane localization signal. Yeast strain AH109 was transformed with prey and bait vector as indicated and plated on to SD medium lacking leucine and tryptophan (-L/-T) or lacking leucine, tryptophan, histidine and adenine (-L/-T/-H/-A). Growth on the latter indicates an interaction between bait and prey. (D) A Model for interactions of Cdc42, RacA with components of Nox complex.
Fig 3.
Bimolecular fluorescence complementation analysis of interaction between BemA, NoxR, Cdc42 and RacA in Epichloë festucae.
BemA, NoxR, Cdc42 and RacA tagged with nGFP (2–174) or cGFP (175–239), were expressed in E. festucae hyphae under the control of their native promoters as indicated. GFP fluorescence was observed at hyphal tips (A) or sites of hyphal cell fusion (B). Arrowheads indicate hyphal fusions. Bars = 5 μm.
Fig 4.
Active form of RacA and Cdc42 can interact with components of fungal Nox complex.
(A) Yeast two-hybrid assays of the interactions between Epichloë festucae constitutive active (CA-) or negative (CN-) form of RacA and Cdc42 with NoxR or BemA. All Rho GTPase derivatives have a mutation (cysteine to alanine) in C-terminal plasma membrane localization signal. Yeast strain AH109 was transformed with prey and bait vector as indicated and plated on to SD medium lacking leucine and tryptophan (-L/-T) or lacking leucine, tryptophan, histidine and adenine (-L/-T/-H/-A). Growth on the latter indicates an interaction between bait and prey. (B, C) Subcellular localization of CA- or CN- form of RacA and Cdc42 in E. festucae hyphae. GFP-tagged RacA or Cdc42 were expressed in E. festucae under the control of the Tef promoter. Bars = 5 μm.
Fig 5.
Overlapping and distinct pheynotypes of Epichloë festucae cdc42 and racA mutants in axenic culture.
(A) Colony morphology of E. festucae wild type (WT), cdc42 and racA mutants on PDA grown for 12 days. (B) Colony diameter, hyphal branch, hyphal diameter and septa formation of E. festucae WT, cdc42 and racA mutants grown on PDA for 12 days. Data are means ± standard error. n = 10 for colony diameter, n = 20 for hyphal branch, hyphal diameter and septa formation. (C) Hyphal tip growth of WT, cdc42 and racA mutant on water ager. Hyphae of endophyte strains were stained with calcofluor white and monitored with confocal laser microscopy. Bars = 10 μm. (D) Hyphal fusion of E. festucae WT, cdc42 and racA mutants grown on water agar. (left) E. festucae strains were grown on water agar for 12 days, stained with calcofluor white and monitored with confocal laser microscopy. Arrowheads indicate hyphal fusions. Bars = 10 μm. (right) The number of hyphal fusions of E. festucae strains grown on water agar were counted using a fluorescence microscope after staining with calcofluor white. Data are means ± standard error from 30 sites from three colonies of each strain. Data marked with asterisks are significantly different as assessed by two-tailed Student’s t tests: **P < 0.01.
Fig 6.
Reactive oxygen species production of Epichloë festucae wild type, racA and cdc42 mutants.
(A) (left) Light micrographs showing production of reactive oxygen species (ROS), as detected by nitroblue tetrazolium (NBT) staining, in E. festucae WT, racA (ΔracA) and cdc42 mutant (Δcdc42) mutant. Hyphal tips stained with NBT were shown by arrow heads. Bar = 20 μm. (right) Frequency of NBT-stained hyphal tips of wild-type, racA mutant and cdc42 mutant. Numbers in column indicate hyphal tips counted. E. festucae strains were grown on PDA for 7 days and stained with NBT for 6 hours. Data marked with asterisks are significantly different as assessed by one-tailed Mann-Whitney U tests: **P < 0.01. (B) L-012-mediated detection of ROS production by E. festucae WT, racA mutant and cdc42 mutant. Colony edge of endophyte strains grown on PDA was treated with L-012 and ROS production was detected as chemiluminescence (top). Value of chemiluminescence relative to wild type was scored. Data are means ± standard error. n = 179. Data marked with asterisks are significantly different as assessed by two-tailed Student’s t tests: **P < 0.01.
Fig 7.
Cdc42 is essential for systemic infection of Epichloë festucae in perennial ryegrass.
(A) Perennial ryegrass was inoculated with E. festucae wild type (WT), racA mutant (ΔracA) or cdc42 mutant (Δcdc42) expressing GFP. Infection of endophyte strain in top, middle and bottom part of leaf blade and pseudostem of host plant was monitored by confocal microscopy. Bars = 50 μm. (B) Biomasses of E. festucae in perennial ryegrass leaves were determined by quantitative PCR 2 month after inoculation as relative amount of the endophyte Ef-ldtE gene to that of perennial ryegrass Lp-act gene. Data are means ± standard error (n = 6 for WT and n = 3 for cdc42 and racA mutants). Data marked with asterisks are significantly different from control (biomass of wild type in corresponding plant tissues) as assessed by the two-tailed Student’s t test: *P < 0.05 and ** 0.01.
Fig 8.
cdc42 mutant can systemically colonize young tissues of perennial ryegrass.
(A) Lateral bud of perennial ryegrass infected with Epichloë festucae wild type (WT), cdc42 mutant (Δcdc42) and racA mutant (ΔracA) expressing GFP. Bars = 100 μm. (B) cdc42 mutant can systemically infect young tillers of perennial ryegrass. Bars = 50 μm.
Fig 9.
Identification of essential amino acids for specific interactions between Cdc42-BemA or RacA-NoxR.
(A) Alignment of the deduced amino acid sequences for Epichloë festucae Cdc42 and RacA. Conserved domains among Rho GTPases are boxed. Amino acids specifically conserved among fungal Cdc42 and RacA are indicated by blue and red letters, respectively. (B) Yeast two-hybrid assays of the interactions between E. festucae NoxR and chimeric or mutated Cdc42 and RacA. (C) Yeast two-hybrid assays of the interactions between E. festucae BemA and chimeric or mutated Cdc42 and RacA. Rho GTPases have mutation in C-terminal plasma membrane localization signal. Yeast strain AH109 was transformed with prey and baid vector as indicated and plated on to SD medium lacking leucine and tryptophan (-L/-T) or lacking leucine, tryptophan, histidine and adenine (-L/-T/-H/-A). Growth on the latter indicates an interaction between bait and prey.
Fig 10.
Binding of RacA/Cdc42 to NoxR is required for hyphal fusion and symbiotic infection of Epichloë festucae.
(A) Colony morphology and diameter of E. festucae wild type (WT), racA mutant and complemented strains on PDA grown for 10 days. Data are means ± standard error. n = 9. Data marked with asterisks are significantly different from wild type as assessed by two-tailed Student’s t tests: **P < 0.01. (B) Hyphal tip growth of WT, racA mutant and complemented strains on PD ager for 20 days. Bars = 20 μm. (C) L-012-mediated detection of ROS production by E. festucae WT, racA mutant and complemented strains. Colony edge of endophyte strains grown on PDA was treated with L-012 and ROS production was detected as chemiluminescence. Value of chemiluminescence relative to wild type was scored. Data are means ± standard error. n = 18. Data marked with asterisks are significantly different from wild type as assessed by two-tailed Student’s t tests: **P < 0.01, *P < 0.05. (D) Hyphal fusion of E. festucae WT, racA mutant and complemented strains grown on water (top) E. festucae strains were grown on water agar for 10 days, stained with calcofluor white and monitored with fluorescence microscopy. Arrowheads indicate hyphal fusions. Bars = 10 μm. (bottom) The number of hyphal fusions of E. festucae strains grown on water agar. Data are means ± standard error from 30 sites from three colonies of each strain. (E) Phenotype of perennial ryegrass infected with E. festucae WT, racA mutant or complemented strains. Photographs were taken approx. 11 weeks after inoculation.
Fig 11.
Binding of RacA/Cdc42 to BemA is required for hyphal polarized growth, but not for host infection of endophyte.
(A) Hyphal tip growth of Epichloë festucae wild type (WT), cdc42 and complemented strains on water ager. Hyphae of endophyte strains were stained with calcofluor white and monitored with confocal laser microscopy. Bars = 10 μm. (B) Colony morphology (left) and diameter (right) of E. festucae WT, cdc42 mutant and complemented strains on PDA grown supplemented 0.01% SDS for 12 days. Data are means ± standard error. n = 5. Data marked with asterisks are significantly different from wild type as assessed by two-tailed Student’s t tests: **P < 0.01, *P < 0.05. (C) Colonization of E. festucae WT, cdc42 mutant and complemented strains in top part of perennial ryegrass tillers approx. 2 months after inoculation. Hyphae (blue lines) and septa (green dots) were visualized by WG-AF488/aniline blue staining monitored by confocal microscopy. Bars = 40 μm. (D) Semi-quantitative PCR detection of endophyte biomass in top part of perennial ryegrass tillers. Specific primes for E. festucae b-tubulin (Ef-Tub) and perennial ryegrass Actin gene (Lp-Act) were used for detection of endophyte biomass and internal standard, respectively.
Fig 12.
Summary of functions of Epichloë festucae Cdc42 and RacA.
(A) Phenotype of racA and cdc42 mutants indicated that RacA is involved in the regulation of growth pattern and cell-cell fusion of E. festucae hyphae, whereas Cdc42 is required for in intercalary hyphal growth in expanding host leaves. Hyphae of the endophyte in host plant are drawn as light blue lines in top diagram. Bars = 50 μm. (B) Summary of interactions between factors in Nox complex and functions of Cdc42 and RacA. Factors in parentheses indicate the interacting partner required for each function of small GTPases.