Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A. oryzae Aohex1 gene homologous to Neurospora crassa HEX1 gene encoding a major protein in Woronin body was expressed as a fusion with DsRed2, resulting in visualization of Woronin body. Confocal microscopy and three-dimensional reconstruction of images visualized the septal pore as a dark region surrounded by green fluorescence of EGFP-fused secretory protein, RNase T1, on the septum. Dual fluorescent labeling revealed the plugging of the septal pores adjacent to the lysed apical compartments by Woronin bodies during hypotonic shock. Disruption of Aohex1 gene caused disappearance of Woronin bodies and the defect to prevent extensive loss of cytoplasm during hypotonic shock.     

Woronin bodies,their impact on stress resistance and virulence of the pathogenic mould Aspergillus fumigatus and their anchoring at the septal pore of filamentous Ascomycota

Hyphae of filamentous Ascomycota consist of compartments that are connected via septal pores. To avoid a dramatic loss of cellular content after wounding, fungi developed mechanisms to occlude their septal pores. In most Pezizomycotina, so‐called Woronin bodies are anchored in proximity to the pore. This is a prominent example for precise spatial positioning of organelles, but so far the underlying molecular organization has remained largely unknown. Using the pathogenic mould Aspergillus fumigatus, we provide evidence that Woronin bodies are important for stress resistance and virulence. Furthermore the molecular machinery anchoring them at the septum is described. Namely, we have identified Lah as the tethering protein and provide evidence that the Woronin body protein HexA binds to the septal pore in a Lah‐dependent manner. Moreover, we demonstrate that a striking poly‐histidine motif targets HexA to the septal cell wall. Thus, the axis HexA‐Lah is an excellent candidate for the tether linking Woronin bodies to the septum. This model applies to A. fumigatus, but most likely also to the vast majority of the Pezizomycotina. Our findings shed light on the evolution of Woronin body anchoring and provide a basis for the development of novel strategies to combat fungal pathogens like A. fumigatus.     

Fungal evo-devo: organelles and multicellular complexity

Peroxisome-derived Woronin bodies of the Ascomycota phyla, and the endoplasmic reticulum (ER)-derived septal pore cap (SPC) of the Basidiomycota, are both fungal organelles that prevent cytoplasmic bleeding when multicellular hyphal filaments are wounded. Analysis of Woronin body constituent proteins suggests that these organelles evolved in part through gene duplication and co-opting of non-essential genes for new functions, indicating that new organelles can arise through typical evolutionary mechanisms. Interestingly, clades possessing the Woronin body and SPC also produce the largest and most complex multicellular fungal reproductive structures. Certain Woronin body and SPC mutants have defects in growth and development, suggesting functions beyond cellular wound healing. I argue that studying these specialized systems will help to reveal the basis for fungal diversity and provide general principles for co-evolution of organelles and multicellular complexity.     

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter‐compartmental and inter‐hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome‐derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild‐type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α‐glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.     

Response of severed Penicillium chrysogenum hyphae following rapid Woronin body plugging of septal pores

Colonies of Penicillium chrysogenum (Thom) Wisconsin strain Q176 were fixed at varying time intervals after having been severed with a razor blade and were examined by transmission electron microscopy. Within such colonies Woronin bodies were found to have plugged septal pores on either side of the cut, both towards and away from the hyphal apices. A very close association of Woronin bodies with septa was retained in damaged compartments emptied of virtually all other contents. In colonies fixed 3 h after cutting, deposition of material over the plugged pores occurred on the side of the septum away from the cut. The newly deposited material was similar in appearance to hyphal wall and septal plate constituents. This consolidation of the seal was apparently completed within 3 h because no further change was observed in colonies fixed 17 h after cutting.     

A Tether for Woronin Body Inheritance Is Associated with Evolutionary Variation in Organelle Positioning

Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB–specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.     

Differential distribution of the endoplasmic reticulum network as visualized by the BipA-EGFP fusion protein in hyphal compartments across the septum of the filamentous fungus, Aspergillus oryzae

We visualized the endoplasmic reticulum (ER) network by expression of the BipA-EGFP fusion protein in the filamentous fungus, Aspergillus oryzae, and focused on the spatial difference of the ER distribution throughout hyphae. The ER formed an interconnected network with motility and displayed a gradient distribution from the apical region. The ER was also found as a tubular network along the septum, which was formed soon after the completion of septation. Discontinuity of the ER network distribution was noticed between the adjacent compartments across the septum, suggesting that the cellular activities in these compartments were independently regulated although they are considered to communicate with each other through the septal pore. Moreover, the ER-visualized strain was subjected to a hypotonic shock, leading to hyphal tip bursting where the Woronin body plugs septal pores and prevents excessive loss of the cytoplasm. In the compartment adjacent to the burst apical tip, the ER network structure and motility were still retained. We also observed re-growth of hyphae from the plugged septa forming intrahyphal hyphae in which the ER network distribution, specialized for apical growth, was regenerated.     

Phosphorylation of the Aspergillus oryzae Woronin body protein, AoHex1, by protein kinase C: evidence for its role in the multimerization and proper localization of the Woronin body protein

Woronin body, a specialized peroxisome, is a unique organelle involved in septal pore sealing and protecting filamentous fungus from excessive cytoplasmic bleeding. We recently characterized the Aohex1 gene encoding the major protein of the Woronin body in the fungus Aspergillus oryzae. Although three-dimensional microscopy revealed plugging of the septal pore by Woronin body, the mechanism of its formation remains unknown. We report here a reduction in the oligomeric forms (dimeric and tetrameric) of AoHex1 upon l-phosphatase treatment, which indicated that AoHex1 phosphorylation in vivo facilitates its oligomerization. Concomitant with the presence of a highly conserved predicted PKC (protein kinase C)-phosphorylatable site (Ser151), the recombinant AoHex1 was phosphorylated by PKC in vitro and the administration of the PKC inhibitors, bisindolylmaleimide I and chelerythrine, resulted in the reduction of the oligomeric forms of AoHex1 in vivo. While spherical dot-like Woronin bodies were visualized by expressing the dsred2-Aohex1 and egfp (enhanced green fluorescent protein)-Aohex1 constructs in A. oryzae, treatment with the PKC inhibitors caused an abnormal localization to ring-like structures. In addition to the reduced phosphorylation of the mutagenized recombinant AoHex1[S151A] (Ser151 to alanine substitution) by PKC in vitro, the overexpression of Aohex1[S151A] as dsred2 fusion against the wild-type background also showed reduction of the oligomeric forms of the endogenous AoHex1 and its perturbed localization to ring-like structures in vivo. In conclusion, the present study implicates the relevance of PKC-dependent phosphorylation of the Woronin body protein, AoHex1, for its multimerization and proper localization.     

Hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores

We have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or Woronin bodies, of Neurospora crassa. Analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. When grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. The HEX-1 protein was predicted to be 19,125 Da. Analysis of the N-terminus of the purified protein indicated that 16 residues are cleaved, yielding a protein of 17,377 Da. A polyclonal antibody raised to the HEX-1 protein recognized multiple forms of the protein, apparently dimers and tetramers that were resistant to solubilization by sodium dodecyl sulfate and reducing reagents. Treatment of the protein with phosphatase caused dissociation of these oligomers. Preparations enriched in Woronin bodies contained catalase activity, which was not detected in comparable fractions from the hex-1(-) mutant strain. These results support the hypothesis that the Woronin body is a specialized peroxisome that functions as a plug for septal pores.     

ATP prevents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici

Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound‐induced cytoplasmic bleeding “flushes” WBs into the septal opening. Alternatively, contraction of septum‐associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane “balloon,” extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1‐enhanced green fluorescent protein (eGFP) appeared associated with the “ballooning” plasma membrane, indicating that cytoplasmic ZtHex1‐eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m‐chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m‐chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1‐eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP‐dependent process.     

Some ultrastructural features ofRhodosporidium toruloides Banno

A 21-day subculture ofRhodosporidium toruloides Banno produced septate mycelium with hook-shaped cells over many septa. In the light microscope these hook-shaped cells appeared to be clamp connections; this feature might suggest thatR. toruloides belongs to the basidiomycetes. Electron micrographs, however, demonstrated that the hyphal septum above which the hook cell occurred and the back septum of the hook cell were tapered centripetally, contained a pore in the middle, and were typical of the ascomycete septum. Two electropaque bodies, believed to be similar to Woronin bodies, were observed occluding the pore in one hyphal septum above which was a hook cell. Melanospores in the 21-day subculture were produced terminally on hyphae and laterally above hook cells which occurred over ascomycete-type septa. Proliferating hook cells appeared similar to repeating croziers found in the ascomycetes. The walls of hyphal cells from the 21-day subculture demonstrated lenticular bodies and alternating electrolucent and electropaque lamellae. The walls of 12-hr dividing yeast cells were observed to be similarly lamellate, a characteristic which has been reported typical of heterobasidiomycetous yeasts. SinceR. toruloides exhibited ascomycetous and basidiomycetous features, a reconsideration of its present classification is suggested. TheRhodosporidium observations are a portion of a thesis submitted by JAJ-R in partial fulfillment of the requirements for the Master of Science degree, North Carolina State University. Supported in part by NIH Grant AI-07122 and USDA Experiment Station Project 5185 to the junior author and by NSF Fellowship GZ 919 to the senior author. To whom grateful acknowledgment is made for use of photographic facilities.     

Budding in the Dimorphic Fungus Phialophora dermatitidis

Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.     

Observation of Woronin bodies in Arthrinium aureum by scanning electron microscopy

Woronin bodies are cytoplasmic organelles of filamentous fungi that can be observed on one, or both sides of each septum. The goal of this paper is to illustrate the presence of them in hyphae of Arthrinium aureum by means of scanning electron microscopy and to show that they act as a safety plug to close septa pores in hypha. Results show that Woronin bodies as an immediate response to prevent a cytoplasm loss. Results support hypothesis proposed previously in literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

SO, a protein involved in hyphal fusion in Neurospora crassa, localizes to septal plugs

The colony of a filamentous ascomycete fungus typically grows as a multinucleate syncytium. While this syncytial organization has developmental advantages, it bears the risk of extensive damage caused by local injury of hyphae. Loss of cytoplasm in injured hyphae is restricted by the fast and efficient sealing of the central pores of hyphal crosswalls, or septa, by a peroxisome-derived organelle called the Woronin body. The formation of septal plugs is also associated with development and leads to separation of certain parts of the colony. Septal plugs associated with developmental processes or aging hyphae typically occur by the accumulation of sealing material. Here we report that in Neurospora crassa, a protein necessary for hyphal fusion and proper colony development called SO (SOFT) localizes to septal plugs. In response to injury, SO accumulates at the septal plug in a Woronin body-independent manner. However, the presence of the Woronin body affects the speed of accumulation of SO at the septal pore. We determined that SO contributes to, but is not essential for, septal plugging. SO accumulation was also observed at septal plugs formed during hyphal aging and during programmed cell death mediated by genetic differences at heterokaryon incompatibility (het) loci.     

Development of woronin bodies from microbodies inFusarium oxysporum f. sp.lycopersici

Summary Woronin bodies are cytoplasmic organelles which commonly lie near the septa in ascomycetous fungi. Although these organelles were observed nearly 100 years ago, little is known about their origin and development. The present ultrastructural investigation describes the ontogeny of Woronin bodies inFusarium oxysporum f. sp.lycopersici [Sacc.] Snyd. and Hans. In this fungus, Woronin bodies are produced by microbodies. Development of the Woronin body begins with the appearance of electron dense material within the microbody. This material aggregates adjacent to the membrane of the microbody and condenses into a single paracrystalline inclusion. Following its formation, the inclusion is gradually extruded and is eventually separated from the parent organelle by an exocytotic mechanism. After the separation, the paracrystalline inclusion is found at the septal pore. Although many recent electron microscopic studies have used various terms to designate these membrane bound organelles, inFusarium these inclusions are believed to correspond to the Woronin bodies initially described by light microscopists.     

Aovps24, a homologue of VPS24, is required for vacuolar formation which could maintain proper growth and development in the filamentous fungus Aspergillus oryzae

Vps24 (vacuolar protein sorting) is a component of ESCRT III (endosomal sorting complex required for transport), which is required for the formation of MVB (multivesicular body). We have isolated the VPS24 homologue gene, Aovps24, from the filamentous fungus Aspergillus oryzae, and analyzed the localization of AoVps24 using EGFP. AoVps24 was localized in the cytoplasm and late endosome-like structures. Furthermore, we constructed an Aovps24 disruptant, which showed impaired growth, conidiation, and hyphal morphology. In addition, normal vacuoles were not observed in the Aovps24 disruptant. In the Saccharomyces cerevisiae vps24 disruptant, the normal vacuoles are formed and it does not show the impaired growth and abnormal cell shape as the A. oryzae Aovps24 disruptant. The results suggest that AoVps24 is required for vacuolar formation and normal vacuoles could have the function to maintain the normal hyphal elongation and conidiation in A. oryzae.     

Role of p54 RNA Helicase Activity and Its C-terminal Domain in Translational Repression, P-body Localization and Assembly

The RNA helicase p54 (DDX6, Dhh1, Me31B, Cgh-1, RCK) is a prototypic component of P-(rocessing) bodies in cells ranging from yeast to human. Previously, we have shown that it is also a component of the large cytoplasmic polyadenylation element-binding protein translation repressor complex in Xenopus oocytes and that when tethered to the 3′ untranslated region, Xp54 represses reporter mRNA translation. Here, we examine the role of the p54 helicase activity in translational repression and in P-body formation. Mutagenesis of conserved p54 helicase motifs activates translation in the tethered function assay, reduces accumulation of p54 in P-bodies in HeLa cells, and inhibits its capacity to assemble P-bodies in p54-depleted cells. Similar results were obtained in four helicase motifs implicated in ATP binding and in coupling ATPase and RNA binding activities. This is accompanied by changes in the interaction of the mutant p54 with the oocyte repressor complex components. Surprisingly, the C-terminal D2 domain alone is sufficient for translational repression and complete accumulation in P-bodies, although it is deficient for P-body assembly. We propose a novel RNA helicase model, in which the D2 domain acts as a protein binding platform and the ATPase/helicase activity allows protein complex remodeling that dictates the balance between repressors and an activator of translation.     

A new self-assembled peroxisomal vesicle required for efficient resealing of the plasma membrane

The Woronin body is a membrane-bound organelle that has been observed in over 50 species of filamentous fungi. However, neither the composition nor the precise function of the Woronin body has yet been determined. Here we purify the Woronin body from Neurospora crassa and isolate Hex1, a new protein containing a consensus sequence known as peroxisome-targeting signal-1 (PTS1). We show that Hex1 is localized to the matrix of the Woronin body by immunoelectron microscopy, and that a green fluorescent protein- (GFP-)Hex1 fusion protein is targeted to yeast peroxisomes in a PTS1- and peroxin-dependent manner. The expression of the HEX1 gene in yeast generates hexagonal vesicles that are morphologically similar to the native Woronin body, implying a Hex1-encoded mechanism of Woronin-body assembly. Deletion of HEX1 in N. crassa eliminates Woronin bodies from the cytoplasm and results in hyphae that exhibit a cytoplasmic-bleeding phenotype in response to cell lysis. Our results show that the Woronin body represents a new category of peroxisome with a function in the maintenance of cellular integrity.