Association of N. crassa with Scots pine

In order to investigate the alternative lifestyles of N. crassa, Scots pine seedlings grown in microcosm were inoculated with conidial suspension (105 conidia/ml) and the colonization patterns were documented over a period of 5 months by fluorescence and confocal microscopy (Supplementary Fig. S1). Most of seedlings looked healthy and were indistinguishable from those without inoculation. Surprisingly, however, fungal hyphae expressing GFP were observed from inside of inoculated seedlings, but not from uninoculated ones. During its growth in the cells of Scots pine seedlings, N. crassa was found to proliferate and survive for up to 5 months without causing any disease symptoms, suggesting its endophytic lifestyle. Inside the roots, fungal growth was confined mostly to the root epidermis and cortex layers (Supplementary Fig. S1e). More compelling evidence of endophytic lifestyle is being described in the later section. To further decipher the innate association between N. crassa and Scots pine, we performed a series of inoculation experiments on Scots pine seedlings grown on water agar.

Can N. crassa be a plant pathogen on Scots pine?

Our most remarkable finding is that N. crassa can act not only as an endophyte but also as a pathogen on Scots pine, when the host plant was grown on water agar or under controlled environments in the greenhouse. Infection with N. crassa incited typical disease symptoms, eventually causing the death of Scots pine seedlings. The mortality rate reached to 83% (90 out of 108 seedlings) at 5 weeks post inoculation (wpi) (Fig. 1a). The abiosis of Scots pine caused by N. crassa takes 4–5 weeks, whereas its well-adapted fungal pathogen Heterobasidion annosum exerts a similar effect in only 3–4 weeks (96% mortality rate) (Fig. 1b). During the initial stage of infection, N. crassa conidia germinated, formed a hyphopodia-like structure (Supplementary Fig. S2c), penetrated into plant tissues and grew intra- or intercellularly between adjacent cells (Fig. 1d–i; Supplementary Fig. S2d–f; Supplementary Fig. S3). Invasive growth continued from root cortical cells (Fig 1d–e) to the core area (Fig. 1f) and they were found in almost 50% of infected root cells at 5 wpi (Supplementary Fig. S4). At the end of the infection stage, N. crassa hyphae could grow out from the stomata on the stem of infected Scots pine seedling (Supplementary Fig. S5a–f) and develop conidiophores with conidia (Supplementary Fig. S5g). These observations clearly demonstrate that N. crassa can complete its life cycle in association with Scots pine and further support the hypothesis that N. crassa has a pathogenic lifestyle. Moreover, culture filtrate of N. crassa induced similar cell death in Scots pine seedlings (Supplementary Fig. S6b), suggesting that N. crassa may produce phytotoxic compounds and function as a necrotrophic plant pathogen on Scots pine. To understand if N. crass can incite disease symptom not only in seedlings grown on water agar, but also grown trees, 3-year-old Scots pine trees were inoculated with wood dowels pre-colonized by N. crassa in the greenhouse. N. crassa could incite clear necrosis on 3-year-old trees 6 wpi. The necrosis areas by N. crassa and H. annosum were 42.5% and 67.2%, respectively, when measured by the ratio of the white (healthy) and brown area (necrosis) (Fig. 2a–c). These infected trees were sampled and heat-treated at 121°C for 10 min to understand whether N. crassa inside of tree could survive under harsh conditions such as forest fire. Surprisingly, N. crassa was grown out and was the sole surviving fungal taxon on wood trunks after heat treatment when incubated for 2 weeks (Fig. 2d). This further supports our previous finding and suggests that N. crassa within host cells can survive as a pathogen or an endophyte and grow out from the burned tree as a saprotroph.

Figure 1 Pathogenic interactions between N. crassa and Scots pine seedlings. Scots pine seedlings were inoculated with N. crassa (a), H. annosum (b) and control (c). (d) Transverse section of Scots pine root inoculated with N. crassa. Plant cell walls were stained with PI and fungal hyphae were labeled with WGA. (e and f) Transverse sections of Scots pine roots inoculated with N. crassa FGSC 10589. GFP images were obtained by staining with FM4-64 at different stages of infection from 3 (e) to 5 (f) weeks post inoculation (wpi). (g) Image of N. crassa hyphae stained with WGA within host plant cells. (h) SEM image of N. crassa hyphae growing from one plant cell to another. (i) Colored SEM image, red and green indicate plant cell wall and N. crassa hyphae, respectively. Bars = 1 cm (a–c); 100 μm (d, e and f); 10 μm (g); 5 μm (h). N. crassa strains used in a, d, g, h and i was FGSC 2489. Full size image

Figure 2 Infection of 3-year-old Scots pine trees by N. crassa. Disease symptoms caused by N. crassa (a), H. annosum (b) and control (c). Disease symptom was measured 6 wpi. Red arrows indicate typical necrosis symptoms. (d) Survival of N. crassa in plant tissues after heat treatment. Three-year-old N. crassa-infected trees were heat-treated to 121°C at 100 kPa for 10 min, followed by incubation at 24°C for 2 weeks. (e) Three-year-old Scots pines used for infection experiments in the greenhouse. Bar = 1 cm. Full size image

Biochemical and molecular mechanisms underlying N. crassa and Scots pine interactions

To determine whether N. crassa infection elicits a defense response in Scots pine seedlings, host-defense-related reactions were observed. Plant cells at infection sites killed by N. crassa were observed by staining with Evans blue (Supplementary Fig. S6a). Furthermore, callose deposition and the accumulation of ROS around infection sites on stem were evident following staining with aniline fluorochrome blue (Fig. 3a) and diaminobenzidine (DAB) (Fig. 3b), respectively. These data indicate that the interaction between N. crassa and Scots pine represents a typical host–pathogen interaction.

Figure 3 Host response and gene expression during interaction with N. crassa. (a) Callose deposition around the infection site on stem, stained with aniline fluorochrome. (b) Accumulation of reactive oxygen species (ROS) at the infection site on stem, stained with diaminobenzidine (DAB). Bars = 20 μm. (c) Expression profiles of defense-related genes in the roots of Scots pine after N. crassa inoculation: ACRE, Avr9/Cf-9 rapidly elicited defense-related gene; PER65, peroxidase 65; PSYP1, class III peroxidase; GPX, glutathione peroxidase; DEF1, defensin; and CAT, catalase. (d) Expression profiles of N. crassa genes during its interaction with Scots pine's roots. nip, Necrosis-inducing protein; cat, catalase-1; per, dyp-type peroxidase; oxi-1 and oxi-2, two oxidoreductases. The level of expression was measured at one (T1) and two (T2) weeks after inoculation. Fold changes are relative to uninfected Scots pine seedlings (c) and N. crassa grown on Vogel's medium (d). The bars indicate standard deviation among 3 biological replicates. Full size image

In addition to biochemical response, expression patterns of ROS- and defense-related genes, such as those encoding peroxidases15 (peroxidase 65 [PER65], class III peroxidase [PSYP1] and glutathione peroxidase [GPX]), Avr9/Cf-9 rapidly elicited defense-related gene (ACRE)16, defensin (DEF1)17 and catalase (CAT)15 were monitored by qRT- PCR. All genes were differentially expressed in Scots pine's roots at 2 wpi with N. crassa (Fig. 3c). Expression of genes encoding catalase and peroxidases was most highly up-regulated. Expression of ACRE and DEF1 was also up-regulated in the roots (Fig. 3c) and stems (Supplementary Fig. S7a)after N. crassa infection. We also monitored the expression profiles of pathogenicity-related genes in N. crassa, including those encoding necrosis-inducing protein (nip)18, endoglucanase IV (egl-4)19, catalase (cat), peroxidase (per) and two oxidoreductases (oxi-1 and oxi-2).

Expression of egl-4 was highly up-regulated in roots (Fig. 3d and Supplementary Fig. S7b). Similar expression pattern of egl-4 was observed in H. irregulare during infection of Scots pine19. Expression of the genes encoding the two oxidoreductases and catalase were also highly up-regulated during interaction with Scots pine seedling (Fig. 3d). Together, expressions of genes responsible for ROS modulation were highly up-regulated in both the host and the pathogen. ROS is known to play key roles in maintaining the balance between endophytic and pathogenic fungal lifestyles with host plants20. Reducing ROS levels in the host can stimulate latent pathogens to cause disease20,21. Our gene expression data suggest that the pathogen and the host use opposite strategies to manipulate the level of ROS to maintain their relationships. Scots pine appears to reduce the expression of catalase (leading to accumulation of ROS) (Fig. 3c), resulting in a toxic response to the pathogen22 in the roots. In N. crassa, on the other hand, expression of catalase gene was highly up-regulated at an early stage of root infection (Fig. 3d), reducing the plant-derived ROS level. Later, the host catalase begins to be produced at high levels (Fig. 3c), likely due to excess ROS caused by the presence of the pathogen. Catalase expression of N. crassa was down-regulated, maintaining the virulence (Fig. 3d) of the fungus to the host, which can lead to an ectopic oxidative burst and cell death (accumulation of ROS)23. At the same time, both the pathogen and the host were preparing for the next phase of interactions in the other part of plant such as stem cells (Supplementary Fig. S7a,b). Since expression of a gene encoding a flavoprotein oxidoreductase (NCU06061)24 was highly up-regulated during infection (Fig. 3d), two deletion mutants (Δoxi-1 and Δoxi-2) were tested for their virulence. Indeed, deletion mutants showed significantly lower virulence on Scots pine seedlings (p = 0.02 and 0.07 in Δoxi-1 and Δoxi-2, respectively) (Supplementary Fig. S8), although they did not have any defect on mycological characters including mycelial growth, colony morphology and pigmentation. These combined biochemical and gene expression data suggest that the association between N. crassa and Scots pine is a typical intimate host–pathogen interaction.