I. Multicellularity of Filamentous Fungi

Filamentous fungi form straight hyphae via polarized tubular extension of the hyphal tip and consist of a network of straight primary hyphae with the formation of branches. Hyphae are compartmentalized into distinct cells by the formation of a septum, which is not frequently observed in early-diverging fungi, such as members of the phyla Mucoromycota and Chytridiomycota, but is regularly found in later-diverging fungi belonging to the phyla Ascomycota and Basidiomycota (Jedd 2011). The septum is proposed to increase the mechanical integrity of hyphae and to divide the mycelium into sections of distinct growth states, such as mitotic and non-mitotic cells (Fiddy and Trinci 1976; Momany et al. 2002; Nayak et al. 2010; Edgerton-Morgan and Oakley 2012). However, the septum does not completely separate hyphae due to the presence of a septal pore, which is a perforated structure that allows the exchange of the cytoplasmic constituents, including organelles, between adjacent hyphal cells (Lew 2005; Tey et al. 2005; Ng et al. 2009; Bleichrodt et al. 2015). Such cell-to-cell connectivity resembles gap junctions in animal cells and plasmodesmata in plant cells.

Cell-to-cell connectivity through the septal pore is associated with the catastrophic risk of cytoplasmic loss by cells adjacent to individually damaged hyphae. This risk was demonstrated by the physical damage of hyphae with a razor blade (Trinci and Collinge 1974) or pulse laser (Lichius et al. 2012), treatment with cell wall-destabilizing reagents (Bowman et al. 2002), and exposure to hypotonic shock (Fig. 1a) (Jedd and Chua 2000; Maruyama et al. 2005). Cell death is induced by aging and heterokaryon incompatibility (Fleißner and Glass 2007). Despite such damage, the cytoplasm of cells immediately adjacent to the wounded cell is typically retained (Fig. 1b) (Jedd and Chua 2000; Maruyama et al. 2005). The protected cells then initiate regrowth of the mycelium by producing a new hyphal tip (Jedd and Chua 2000; Maruyama et al. 2006; Maruyama and Kitamoto 2007; Lichius et al. 2012). These processes represent an inherent defense system for promoting survival by preventing the simultaneous loss of cytoplasm from multiple cells upon hyphal wounding. Hence, the septal pore is an important subcellular structure for maintaining the multicellularity of filamentous fungi.

Fig. 1
figure 1

Hyphal wounding and the prevention of excessive cytoplasmic loss. (a) Hyphal tip bursting upon hypotonic shock. Hyphal tips at the margin of an A. oryzae colony grown on agar medium were observed by differential interference contrast (DIC) microscopy before and after flooding hyphae with water. Bar: 50 μm. (b) Prevention of the excessive loss of cytoplasm upon hyphal wounding. The cytoplasm is labeled by expressing EGFP. An arrowhead and arrow indicate a burst hyphal tip and the adjacent septum, respectively. Note that the cell (2nd) adjacent to the lysed cell (1st) retains its cytoplasmic constituents, as determined by DIC and fluorescence microcopy. Bar: 10 μm

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II. The Woronin Body, an Organelle Specific to Pezizomycotina

Filamentous fungal species that form septa have evolved to possess specialized membrane-bound organelles located around the septum; the Pezizomycotina (Ascomycota) contain structures called the Woronin body (Markham and Collinge 1987), and Agaricomycotina (Basidiomycota) have an endoplasmic reticulum (ER)-related septal pore cap (Müller et al. 1998). Woronin bodies were first identified as highly refractive particles located near the septum in the filamentous ascomycete Ascobolus pulcherrimus by the Russian mycologist Michael Stepanovitch Woronin (Woronin 1864). The organelle was later named by Buller (1933) in recognition of Woronin’s discovery and is restricted to Pezizomycotina species (Markham and Collinge 1987). The Woronin body has two morphologically distinct subclasses; it predominantly appears by transmission electron microscopy as a spherical electron-dense structure near the septum (Fig. 2a), although in a small number of species, such as Neurospora crassa, Woronin bodies form hexagonal crystalline structures that are occasionally visible by light microscopy (Fig. 2b) (Markham 1994). By both electron and light microscopy, Woronin bodies were observed to plug the septal pore upon hyphal wounding (Markham and Collinge 1987). Therefore, it was long suspected that the primary function of Woronin bodies was the prevention of excessive cytoplasmic loss from cells adjacent to damaged or lysed cells (Fig. 3a).

Fig. 2
figure 2

Morphology of the Woronin body. (a) Transmission electron microscopic observation of Woronin bodies (arrows) near the septum in A. oryzae. Bar: 500 nm. (b) Differential interference contrast (DIC) image of a hexagonal Woronin body localized to the cell cortex in N. crassa. Woronin bodies are indicated by arrows. Bar: 1 μm

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Fig. 3
figure 3

Woronin body function. (a) Schematic representation of the septal plugging function of Woronin bodies. (b) Confocal images of Woronin bodies (red, arrowheads) and septa (green) before (left) and after (right) hyphal wounding. Woronin bodies and septa were labeled by DsRed2 and EGFP, respectively (Maruyama et al. 2005). Bar: 2 μm

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III. Molecular Basis of Woronin Body Structure

Although Woronin bodies were discovered approximately 150 years ago, the composition of Woronin bodies long remained unclear. Protease digestion of ultrathin sections suggested that Woronin bodies contained proteinaceous materials (McKeen 1971; Mason and Crosse 1975). Woronin bodies from N. crassa were first purified in 2000 through differential and density gradient centrifugation, allowing for the identification of Hex1 as a major structural protein (Jedd and Chua 2000; Tenney et al. 2000). Genes encoding Hex1 are conserved in Pezizomycotina species (Jedd and Chua 2000; Asiegbu et al. 2004; Curach et al. 2004; Soundararajan et al. 2004; Maruyama et al. 2005; Beck and Ebel 2013). Deletion of the hex1 gene results in defective Woronin body formation and severe cytoplasmic bleeding upon hyphal wounding (Jedd and Chua 2000; Maruyama et al. 2005; Beck and Ebel 2013). When the apical neighboring cell is selectively ruptured, the subapical cells reinitiate hyphal growth by producing a new branch, but deletion of the hex1 gene severely reduces or completely loses the activity of growth reinitiation (Tegelaar and Wösten 2017).

Characterization of Hex1 protein revealed that it spontaneously self-assembles to form a solid core, thereby providing mechanical resistance against the protoplasmic streaming pressure that is generated upon hyphal wounding (Jedd and Chua 2000; Yuan et al. 2003). The crystal structure of Hex1 consists of three intermolecular contacts that promote self-assembly (Yuan et al. 2003). Interestingly, the structural properties of Hex1 resemble those of eukaryotic translation initiation factor 5A (eIF5A) proteins (Kim et al. 1998; Peat et al. 1998), suggesting that the hex1 gene evolved from eIF-5A by gene duplication in the ancestor of Pezizomycotina. Phosphorylation of Hex1 also contributes to the formation of the multimeric core of the Woronin body (Tenney et al. 2000; Juvvadi et al. 2007).

The N. crassa hex1 gene encodes a single translational product (Jedd and Chua 2000), although alternative splicing of the hex1 gene was reported in several species, including Magnaporthe grisea, Aspergillus oryzae, and Aspergillus fumigatus (Soundararajan et al. 2004; Maruyama et al. 2005; Beck et al. 2013). Both spliced and non-spliced transcripts yield two polypeptides that sediment in high-density protein fractions (Maruyama et al. 2005), suggesting that these peptides participate in the self-assembly and formation of the Woronin body core matrix. Moreover, it was demonstrated that a poly-histidine motif encoded within an alternatively spliced region targets Hex1 to the septal pore (Beck et al. 2013).

Although the hexagonal crystal structure of the N. crassa Woronin body is easily observed by light microscopy, the fusion of Hex1 with a fluorescent protein enables the septal plugging activity of Woronin bodies to be visualized in most Pezizomycotina species (Fig. 3b) (Maruyama et al. 2005; Beck and Ebel 2013). Using this approach, Bleichrodt et al. (2012) reported that the Woronin body reversibly plugs the septal pore during normal growth, a characteristic that contrasts the conventional view of this organelle functioning in wound healing. The analysis of gene expression activity for individual hyphae revealed that although wild-type cells exhibited heterogeneous activity, cells lacking Woronin bodies through deletion of hex1 (Δhex1 strain) displayed more uniform gene expression activity (Bleichrodt et al. 2012). Thus, it was proposed that Woronin bodies contribute to the generation of hyphal populations with different cellular activities by occasionally preventing cell-to-cell connectivity via the plugging of the septal pore. The generation of such colonial heterogeneity under normal growth conditions may protect cells against environmental stresses, as evidenced by the sensitivity of the A. oryzae Δhex1 strain to heat stress (Bleichrodt et al. 2012). Thus, it appears that Woronin bodies have a gatekeeper role in regulating cell-to-cell channels by simple plugging of septal pore, similar to that observed upon hyphal wounding (Jedd and Pieuchot 2012).

In addition to septal plugging, Hex1 has other physiological impacts at the cellular level. For example, deletion of the hex1 gene results in defective conidiation (asexual spore formation) (Yuan et al. 2003; Son et al. 2013), impaired growth under nitrogen starvation (Soundararajan et al. 2004), and increased sensitivity to cell wall and membrane-destabilizing agents (Beck et al. 2013). In plant pathogenic fungi, Hex1 is also required for efficient pathogenesis and viral RNA accumulation (Soundararajan et al. 2004; Son et al. 2013). These lines of evidence suggest additional physiological functions of Woronin bodies, which need further investigation of the action mechanisms.

IV. Woronin Body Biogenesis from Peroxisomes

Electron microscopic studies examining the subcellular origin of Woronin bodies demonstrated the inclusion of these organelles within microbodies (Wergin 1973; Camp 1977). This finding was supported by the specific binding of antibodies against microbody-specific signal peptides to Woronin bodies (Keller et al. 1991). A relationship between peroxisomes and the Woronin body was clearly indicated by the finding that the C-terminus of Hex1 protein contains peroxisomal targeting signal sequence 1 (PTS1) (Jedd and Chua 2000). In addition, time-lapse imaging analysis revealed that Woronin bodies bud from peroxisomes (Tey et al. 2005).

Woronin body formation occurs at the hyphal apex through a process involving apically biased expression of the hex1 gene (Tey et al. 2005). Woronin body biogenesis requires peroxins that mediate the import of peroxisomal matrix and membrane proteins (Fig. 4a) (Managadze et al. 2007; Liu et al. 2008; Li et al. 2014). Specifically, Hex1 associates with Pex26, a peroxin that is enriched in Woronin body-containing peroxisomes and recruits the AAA ATPases Pex1 and Pex6 to the peroxisomal membrane for receptor recycling (Liu et al. 2011). The recruitment of Pex26 leads to the formation of a parallel activation loop for Pex5 recycling and Hex1 import, allowing for the efficient biogenesis of Woronin bodies. Fam1 was originally identified in Colletotrichum orbiculare as a Pezizomycotina-specific ortholog of Pex22, which functions in the recycling of PTS receptors from peroxisomes to the cytosol (Fig. 4a). Fam1 is specifically localized on the membrane of Woronin bodies, raising the possibility that recycling of PTS receptor occurs in Woronin bodies (Kubo et al. 2015).

Fig. 4
figure 4

Woronin body biogenesis from peroxisomes. (a) Schematic diagrams of the import machineries of peroxisomal matrix and membrane proteins. Peroxins labeled in blue were reported to be involved in Woronin body function. PTS1 peroxisomal targeting signal 1, mPTS membrane peroxisome targeting signal. (b) Model of Woronin body biogenesis from peroxisomes

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In addition, the budding of Woronin bodies from peroxisomes appears to require dynamin-related proteins (Würtz et al. 2008). The peroxisome proliferator protein Pex11 is needed for the differentiation of Woronin bodies from peroxisomes (Fig. 4b) and also affects the septal plugging function of these organelles (Escaño et al. 2009). The genetic screening of N. crassa mutants defective in Woronin body biogenesis identified several peroxins and the Woronin body sorting complex (WSC) protein that were critical for this process (Liu et al. 2008). WSC is a Pezizomycotina-specific protein that recruits the Hex1 assembly to the matrix side of the peroxisomal membrane and facilitates budding of the Woronin body (Fig. 4b) (Liu et al. 2008). WSC belongs to the PMP22 (peroxisome membrane protein)/MPV17 (myeloproliferative leukemia virus 17) gene family and is proposed to have evolved to possess new functional properties, including self-assembly and Hex1 binding, from ancestral PMP22 (Jedd 2011).

ApsB, a component of the microtubule-organizing center (MTOC) that has functional peroxisomal targeting signal sequence 2 (PTS2), interacts with Hex1 (Zekert et al. 2010). In addition, TmpL, a transmembrane protein involved in redox-related signal transduction, is localized to Woronin bodies (Kim et al. 2009). However, it is unclear how ApsB and TmpL are involved in Woronin body biogenesis and function.

V. Septal Tethering of the Woronin body

In most Pezizomycotina species, Woronin bodies are typically tethered to the septum by a filament at a distance of 100–200 nm (Momany et al. 2002; Maruyama et al. 2005). The tethering structure was shown to have elastic properties, as demonstrated by the rapid return of the Woronin bodies to the septum after physical separation by laser trapping (Berns et al. 1992). Moreover, Woronin bodies rapidly insert into the septal pore upon hyphal wounding and also reversibly plug the septal pore during normal growth (Bleichrodt et al. 2012). These processes require the proper positioning of the Woronin body and sufficient flexibility of the tethering linker.

In contrast, the Woronin bodies of a few members of the genera Neurospora and Sordaria are associated with the cortex in a delocalized pattern (Plamann 2009). In N. crassa, newly synthesized Hex1 proteins are imported into peroxisomes in the apical compartment (Tey et al. 2005), and the newly formed Woronin bodies are then inherited into subapical compartments via association with the cell cortex (Tey et al. 2005; Liu et al. 2008). During a screening for mutants that accumulate Woronin bodies in the apical compartment, Ng et al. (2009) identified the leashin locus, which is comprised of the adjacent genes lah-1 and lah-2. The N-terminal region of LAH-1 binds to Woronin bodies via the membrane protein WSC, whereas the C-terminal region mediates the association of Woronin bodies with the cell cortex (Fig. 5a) (Ng et al. 2009). In contrast, LAH-2, which localizes to the hyphal tip and septum, is not involved in Woronin body function (Fig. 5a). In N. crassa cells expressing an LAH-1/LAH2 fusion construct, Woronin bodies accumulate at both sides of the septum, which only has partial ability to prevent the excessive loss of cytoplasm upon hyphal injury (Ng et al. 2009). This finding indicates an additional requirement of the tethered Woronin bodies for septal pore plugging.

Fig. 5
figure 5

Subcellular localization of Woronin bodies and tethering proteins. (a) Delocalized pattern of cortex-associated Woronin bodies via LAH-1 protein. (b) Septal tethering of Woronin bodies via LAH protein. (c) Illustration of the generation of two LAH proteins from the ancestral LAH protein

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Other species, such as A. fumigatus and A. oryzae, with tethered Woronin bodies have large LAH proteins consisting of a single polypeptide of over 5000 amino acids (Beck et al. 2013; Han et al. 2014). LAH is required for the tethering of Woronin bodies to the septum and is involved in the Woronin body function of preventing the excessive loss of cytoplasm but to a lesser extent than the major Woronin body protein Hex1 (Han et al. 2014). This property may be explained by the fact that untethered Woronin bodies are able to plug the septal pore, but not as quickly as tethered ones.

Large LAH protein can be functionally divided into conserved N- and C-terminal regions and a non-conserved central region (Beck et al. 2013; Han et al. 2014): The N-terminal region associates with Woronin bodies in a WSC-dependent manner, and the C-terminal region containing a transmembrane spanning region mediates localization of LAH to the septum (Fig. 5b) (Beck et al. 2013; Han et al. 2014; Leonhardt et al. 2017). A truncated LAH protein consisting of only the N- and C-terminal regions retains the ability to tether Woronin bodies to the septum; however, the Woronin bodies are located closer (~50 nm) to the septum than those in wild-type cells (Han et al. 2014). This difference is roughly consistent with the 70-nm length of the approximately 2700-amino-acid central region of LAH, as was estimated based on the length (1 μm) of the 4-mDa protein titin (Nave et al. 1989). The elasticity of the Woronin body tether that was previously demonstrated by laser-capture experiments (Berns et al. 1992) is conferred by the central non-conserved region of LAH (Han et al. 2014). The non-conserved central region in LAH is predicted to be disordered (Han et al. 2014) and presumably functions as a molecular spring, similarly to the muscle protein titin, which exhibits molecular spring-like elasticity via its intrinsically disordered region (Li et al. 2001). Moreover, in cells expressing LAH protein lacking the central region, tethered Woronin bodies do not plug the septal pore, even after hyphal wounding (Han et al. 2014). Collectively, efficient septal plugging requires not only that Woronin bodies are tethered to the septum but also that the tether must have sufficient elasticity to allow for the relatively unrestricted movement of Woronin bodies. The occasional observation that Woronin bodies from the ruptured cell side plug the septal pore suggests an active mechanism of Woronin body movement (Steinberg et al. 2017a). Surprisingly, ATP depletion by the respiration inhibitor carbonyl cyanide m-chlorophenylhydrazine induces the translocation of Woronin bodies into the septal pore without cell wounding. This suggests that ATP is required to prevent the septal plugging by Woronin bodies, leading to the speculation that ATP may bind to the tethering protein LAH for preventing a conformational change and contraction of the protein (Steinberg et al. 2017b).

The molecular origin of LAH remains elusive, but this protein first appeared in the clade of Pezizomycotina species together with Woronin bodies (Jedd 2011). The ancestral lah gene encodes a single, large polypeptide and was subsequently split into two genes, lah1 and lah2, in the Neurospora-Sordaria clade. The lah1 gene acquired an early termination sequence and a new cortex association domain at the C-terminus and a new promoter controlled expression of the downstream lah2 gene (Fig. 5c). Thus, as a result of the gene splitting of the lah gene, the localization of the Woronin body was shifted from septal tethering to the cortex. N. crassa and Sordaria fimicola exhibit extensive cytoplasmic streaming through septal pores (Lew 2005; Tey et al. 2005; Ng et al. 2009) and have unusually rapid growth rates (>1 μm/s) (Ryan et al. 1943). As the tethering of Woronin bodies to the septum may have reduced the exchange of cytoplasm between adjacent cells, a delocalized pattern of Woronin bodies with cortex association may have been selected to support the rapid growth of these species.

VI. Proteins Associated with the Woronin Body

Through the purification of Woronin body-associated proteins and bioinformatics approaches for detecting proteins that contain intrinsically disordered regions, 17 septal pore-associated (SPA) proteins were identified in N. crassa (Lai et al. 2012). Intrinsically disordered proteins range from partially to completely unstructured and lacking globular folds; however, these proteins are capable of folding upon binding to target molecules (Wright and Dyson 2009). The number of reports related to intrinsically disordered proteins has increased significantly in recent years, and many biological functions of these proteins have been revealed (Oldfield and Dunker 2014). The loss-of-function of several SPA proteins leads to excessive septation, septal pore degeneration, and uncontrolled Woronin body activation (Lai et al. 2012). Spa10 protein is required for the localization of LAH C-terminal region at the mature septal pore, consequently stable tethering of Woronin bodies to the septum (Leonhardt et al. 2017). These findings suggest that the septal pore is a complex subcellular site for the assembly of unstructured proteins, which contribute to diverse states of cell-to-cell connectivity.

The Pezizomycotina-specific protein SO, which was originally found in N. crassa and was named based on the “soft” appearance of the mutant colony, is important for hyphal fusion and sexual reproduction (Fleißner et al. 2005; Engh et al. 2007). SO protein consists of approximately 1200 amino acids and contains a single WW domain. Pro40, a Sordaria macrospora SO homolog, functions as a scaffold by associating with protein kinases involved in cell wall integrity (Teichert et al. 2014). SO protein is dispersed throughout the cytoplasm under normal growth conditions but accumulates at the septal pore adjacent to wounded cells (Fig. 6), as was reported in N. crassa and A. oryzae (Fleißner and Glass 2007; Maruyama et al. 2010). S. macrospora Pro40 protein colocalizes with Woronin bodies (Engh et al. 2007). Deletion of the so gene delays septal plugging and reduces the number of hyphae that prevent excessive cytoplasmic loss in N. crassa and A. oryzae (Fleißner and Glass 2007; Maruyama et al. 2010). Taken together, these findings indicate that SO protein has septal plugging activity by accumulating at the septal pore, similar to the function of Woronin bodies. Additionally, SO protein accumulates at the septal pore in aging hyphae (Fleißner and Glass 2007) and under various stress conditions, including low/high temperature, high acidity/alkalinity, and nitrogen/carbon depletion (Fig. 6) (Maruyama et al. 2010). In response to pulse laser treatment, which physically stresses cells without causing hyphal wounding, SO rapidly accumulates at the septal pore nearest to the stressed hyphal area (Maruyama et al. 2010). Thus, SO protein may regulate cell-to-cell connectivity via the septal pore in a stress-dependent manner.

A study of Aspergillus nidulans revealed that mitosis interrupts cell-to-cell connectivity through the septal pore (Shen et al. 2014). The mitotic NIMA kinase was found to be localized to the septum during interphase and to contribute to keeping the septal pore open but is translocated into the nucleus to initiate mitosis, resulting in septal closure. Notably, however, the mitotic regulation of cell-to-cell connectivity is independent of Woronin bodies and SO protein. Identification of the NIMA kinase substrate(s) may provide insight into the molecular mechanisms underlying mitotic interruption of cell-to-cell connectivity.

Fig. 6
figure 6

Accumulation of SO protein at the septal pore. SO localization was visualized by expressing as EGFP fusion protein. (Left) Upon hyphal wounding, SO accumulates at the septal pore together with the Woronin body. (Right) SO also accumulates at the septal pore under stressed conditions. Asterisk indicates the septum, and the arrowhead indicates the accumulation of SO protein at the septal pore. Bars: 5 μm

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VII. Conclusions and Perspectives

Despite the discovery of Woronin bodies over 150 years ago, our understanding of these unique fungal organelles was exclusively based on primitive microscopic studies conducted in the twentieth century. However, in the past 20 years, molecular structure and function of Woronin bodies have been largely elucidated. Genomic changes such as gene duplication and splitting generated and modified the Woronin body to allow for specific multicellular organization of individual Pezizomycotina species. Although the primary function of Woronin bodies had long been thought to be wound healing, recent molecular studies have revealed new physiological functions of these organelles. To date, however, the molecular machineries controlling and mediating septal closure and cell repair remain mostly unknown. Further investigations to functionally link Woronin bodies and related molecules in the vicinity of the septum will lead to a more comprehensive understanding of the mechanisms regulating cell-to-cell connectivity and fungal multicellularity.