Introduction

A fungal mycelium is the result of fusing hyphae that grow at their apices and that branch subapically. In general, hyphae of the lower fungi, i.e. the Glomeromycota, Zygomycota, and Chytridiomycota are sparsely, if at all, septated [1]–[3]. Therefore, the cytoplasm within mycelia of these fungi is continuous. Hyphae of the higher fungi, i.e. the Ascomycota and Basidiomycota, are compartmentalized by septa. These septa contain central pores of up to 500 nm that allow streaming of cytoplasm and translocation of organelles like mitochondria and nuclei [1]–[3]. Therefore, the cytoplasm within these mycelia is also considered to be continuous. This discriminates the filamentous fungi from plants and animals. In these latter two kingdoms there are also intercellular cytoplasmic connections but they are much smaller. Gap junctions in animals and plasmodesmata in plants have pores with a diameter of about 1.5 to 3.0 nm. These pores allow streaming of inorganic ions and small water-soluble organic molecules [8]–[10]. It should be noted that the diameter of the pores of plasmodesmata and gap junctions is dynamic. For instance, the channels in plasmodesmata can be closed or their width increased to 5 to 9 nm.

The major groups of fungi within the Basidiomycota contain different types of septa. The Pucciniomycotina and the Ustilaginomycotina have relatively simple septa [1], [11], [12]. In contrast, septa of the Agaricomycotina are relatively complex. They consist of a barrel-shaped swelling around the pore, the dolipore, which is associated with a septal pore cap (SPC) [6]. This septal pore cap, which restricts organelle translocation, can be of the vesiculate type, the perforate type or the imperforate type [13] and is assumed to be derived from the endoplasmic reticulum [14]–[16]. The SPC of Schizophyllum commune is of the perforate type. Its base, i.e. the part closest to the septum, has a diameter of 450–600 nm and the whole structure is regularly perforated by openings of approximately 100 nm [15], [17].

Septa of Ascomycota and the Basidiomycetes become plugged in response to hyphal damage to prevent loss of cytoplasm [18]–[20]. Peroxisome-like organelles, called Woronin bodies, plug the septa of the ascomycetes [21]–[23], whereas in basidiomycetes septa are closed by electron dense, plugging material [19]. It has been proposed that the SPC is involved in the plugging process [15], [20], [24], [25]. Here, it is shown by laser dissection that septa of growing hyphae of S. commune not only plug in response to hyphal damage but that this is also caused by environmental conditions such as availability of carbon source, exposure to high temperature, osmotic shock or toxic agents. The results thus imply that the cytoplasmic continuity of this fungus depends on the environmental conditions.

Results and Discussion

S. commune was grown in a glass bottom culture dish in a thin layer of solidified minimal medium (MM) containing glucose as a carbon source. The solidified medium was overlaid with liquid MM (Figure 1A). Extension of selected hyphae was followed during a 3 min period using the light microscope of the PALM CombiSystem. Compartments of growing hyphae were disrupted with the laser of the PALM Combi-system within 30 µm from the septum. Cytoplasm of disrupted compartments spilled into the surrounding medium (Figure 2). Cytoplasmic flow from the adjacent compartment into the medium depended on the state of the septum (Figure 2; Movies S1, S2, S3, S4, S5, S6). Loss of cytoplasm was considerable (septum was open and was slowly closed; Movies S5-S6), minor (septum was open but was quickly closed; Movies S3-S4) or not detected (septum was already closed; Movies S1-S2). By cutting the second compartment and following cytoplasmic streaming from the apical compartment into the medium it was shown that 45 out of 45 apical septa were open (Table 1). In contrast, only 25 out of 53 of the second septum (separating compartment 2 and 3; see Figure 1C) and 1 out of 14 of the third septum (separating compartment 3 and 4) were open. This was shown to occur in growing hyphae throughout the mycelium. The second and third septa closed quickly in the case they were open. In contrast, closure of the most apical septum was generally slower.

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Figure 1. Glass bottom culture dish used to assess plugging of septa in S. commune.

In most cases S.commune was grown in a thin layer of agar medium overlaid with minimal medium (A). However, liquid medium was not added when it was assessed whether septa were open or closed in the absence of glucose in the medium. In this case, the culture dish contained distinct patches of minimal medium with or without glucose, which were separated by a gap of 5 mm (B). (C) Magnification of boxed area in (A) and (B) showing a hypha with the first and second septum as referred to in the text.

https://doi.org/10.1371/journal.pone.0005977.g001

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Figure 2. A septum is open when cytoplasm moves through the septum after laser dissection of a neighbouring compartment.

Compartments before (A, E) and after (B–D, F–H) dissection of a hypha with a closed (A–D) and an open (E–H) septum. Cytoplasmic flow in E–H is indicated by the movement of a vacuole (arrows) through the septum (*). Dots represent the position of the laser.

https://doi.org/10.1371/journal.pone.0005977.g002

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Table 1. Plugging of a septal pore depends on its position in the hypha and the presence of glucose in the medium.

https://doi.org/10.1371/journal.pone.0005977.t001

The fact that the second and third septa were often closed came to a surprise considering the phenomenon of streaming of cytosol in a fungal mycelium [26]. We argued that in the presence of a surplus of glucose in the medium cytosolic streaming is not essential and that therefore septal pores can be closed. To test this hypothesis, hyphae were grown from a glucose-containing medium into a medium without this carbon source (Figure 1B). Indeed, in MM without glucose many more septa of growing hyphae were open (i.e. 11 out of 12 apical septa, 22 out of 29 second septa and 26 out of 41 third septa; Table 1). Taken together, these findings show that the continuity of the cytoplasm of S. commune depends on the presence of carbon source in the medium.

We reasoned that another environmental condition that could affect the septal pore is exposure to stress. To test this, the liquid medium overlaying the solid minimal medium (Figure 1A) was replaced by deionized water. This neither affected growth nor closure of the apical septum in a 60 minutes interval (Table 2). In contrast, addition of 1 M MgSO₄ to the liquid medium did have an effect on the state of the septal pore. Addition of MgSO₄ initially resulted in the accumulation of vacuoles and after 15 minutes most hyphae had stopped growing. At this point, all apical septa were still open. However, 5 out of 5 apical septa had closed after an additional 30 minutes of exposure to 1M MgSO₄. Absence of streaming of cytoplasm into the medium from the adjacent compartment was not due to the presence of vacuoles near the septum. Exposure of S. commune to 20 µg ml⁻¹ nourseothricin, which inhibits protein synthesis, triggered a similar response as 1M MgSO₄ (Table 2). Within 30 minutes, the hyphal tips stopped growing and mild vacuolization was observed. Yet, septa were still open. After another 30 minutes, all hyphae were heavily vacuolized and almost all apical septa had closed. Transfer of the mycelium from 25°C to 0 or −20°C did not cause plugging. Hyphae continued their original growth rate when they were placed back at 25°C. Exposing the mycelium to 45°C for 30 minutes stopped growth. Hyphae vacuolized and all apical septa had closed (Table 2). Interestingly, most septa opened again 15 minutes after colonies were placed back at 25°C. During this time, hyphae restored normal growth and vacuolization was decreased to normal levels.

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Table 2. Plugging of the apical septum depends on environmental stress.

https://doi.org/10.1371/journal.pone.0005977.t002

Our data show that hyphae initially accumulate vacuoles upon exposure to stress, after which their growth halts. This is followed by septal closure. Closure of the septum may thus be a final rescue system that is activated to have individual compartments survive the stress condition. Notably, plugging was shown to be reversible, at least when induced by heat. This indicates that septal pores are dynamic structures that can open and close depending on the environmental conditions. Taken together, it is concluded that the cytoplasm of a mycelium of a higher fungus is not continuous per se, as is generally assumed. The cytoplasmic connections within a mycelium of S. commune thus resemble those in plants and animals. Like the septal pore of this basidiomycete, plasmodesmata and gap junctions can reversibly open and shut [8], [27], [28].

Materials and Methods

Supporting Information