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.     

A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position,Movement, and Function of Woronin Bodies in Aspergillus oryzae

The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies.     

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.     

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.     

Multiple modes for gatekeeping at fungal cell‐to‐cell channels

Cell‐to‐cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co‐workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin body‐dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.     

SEPTAL PORE STRUCTURE IN TRICHODERMA SATURNISPORUM

Electron microscopic observations of a previously undescribed ascomycetous septal pore structure are presented and discussed. Hyphal septa and septa at the base of phialides in the hyphomycete, Trichoderma saturnisporum Hammill, developed a membrane-bounded, electron-opaque septal pore body which was fine-structurally similar to Woronin bodies. Within a septal pore body, several electron-transparent layers appeared to develop centripetally from the septal pore rim. The number of layers observed varied from two to about five, with lower numbers being more frequently observed. The electron-transparent layers perhaps functioned as a vinculum, binding the septal pore body in place. Questions about the origin and function of septal pore bodies are discussed.     

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.     

Woronin bodies in arthrinium genus

Woronin bodies are present near all septal pores and in conidia of Arthrinium strains and may regulate cytoplasmic flow in both injured and actively growing healthy colonies. They vary in size and frequency, the central one plugs the septal pores in actively developing colonies and in mature conidia. The septa are thinner in the Woronin-body region.     

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.     

Orbilia ultrastructure, character evolution and phylogeny of Pezizomycotina

Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota. Although Orbiliomycetes is resolved as the most basal lineage in some analyses, molecular support for the node resolving the relationships between the two classes is low and topologies are unstable. We provide ultrastructural evidence to inform the placement of Orbiliomycetes by studying an Orbilia, a member of the only order (Orbiliales) of the class. The truncate ascus apex in the Orbilia is thin-walled except at the margin, and an irregular wall rupture of the apex permits ascospore discharge. Ascus, ascogenous and non-ascogenous hyphae were simple septate, with septal pores plugged by unelaborated electron-dense, non-membranous occlusions. Globose Woronin bodies were located on both sides of the septum. Nuclear division was characterized by the retention of an intact nuclear envelope, and a two-layered disk-shaped spindle pole body. The less differentiated nature of the spore discharge apparatus and septal pore organization supports an earliest diverging position of Orbiliomycetes within the subphylum, while the closed nuclear division and disk-shaped spindle pole body are interpreted as ancestral state characters for Ascomycota.     

SEPTAL PORES IN THE HETEROBASIDIOMYCETIDAE,PUCCINIA GRAMINIS AND P. RECONDITA

The septal pores in uredial mycelium of Puccinia graminis and P. recondita lack the septal swelling and septal pore cap (dolipore-parenthosome configuration) typically associated with the pores of previously investigated Homobasidiomycetidae and the Tremellales among the Heterobasidiomycetidae. The pores in young hyphae of these two species of Puccinia are characterized by the presence of a cytoplasmic matrix which apparently occludes the pore and acts as a plug, thus preventing the migration of organelles from cell to cell. Large vesicles are typically present at the periphery of the pore matrix and the matrix may be very incompletely bounded by a membrane. Nuclei and other cytoplasmic structures migrate from cell to cell through an opening in the septum lateral to the pore. The available evidence indicates that this peripheral gap in the septum results from a breakdown of a portion of an initially complete septum rather than from incomplete septum formation. In addition to the centripetally formed septa, the hyphae of P. graminis and P. recondita are further compartmentalized by shallow infoldings of the lateral wall and limited unilateral septum formation. There is apparent free passage of cellular material between adjacent compartments.     

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.     

Woronin body function in Magnaporthe grisea is essential for efficient pathogenesis and for survival during nitrogen starvation stress

The Woronin body is a peroxisome-derived dense-core vesicle that is specific to several genera of filamentous ascomycetes, where it has been shown to seal septal pores in response to cellular damage. The Hexagonal peroxisome (Hex1) protein was recently identified as a major constituent of the Woronin body and shown to be responsible for self-assembly of the dense core of this organelle. Using a mutation in the Magnaporthe grisea HEX1 ortholog, we define a dual and essential function for Woronin bodies during the pathogenic phase of the rice blast fungus. We show that the Woronin body is initially required for proper development and function of appressoria (infection structures) and subsequently necessary for survival of infectious fungal hyphae during invasive growth and host colonization. Fungal mycelia lacking HEX1 function were unable to survive nitrogen starvation in vitro, suggesting that in planta growth defects are a consequence of the mutant's inability to cope with nutritional stress. Thus, Woronin body function provides the blast fungus with an important defense against the antagonistic and nutrient-limiting environment encountered within the host plant.     

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.     

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.     

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.     

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.     

Ultrastructure of septa in Blastobotrys and Sporothrix

Transmission electron micrographs of septa in Blastobotrys species invariably showed central micropores. Septa of species of Sporothrix, however, exhibited three types of pores: (a) micropores which were central if single, or scattered; (b) central simple pores with Woronin bodies; (c) dolopores. The results confirm the heterogeneity of the genus Sporothrix.