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.     

Mapping Woronin body position in Aspergillus nidulans

The positions of all Woronin bodies in five germlings of Aspergillus nidulans prepared by plunge freezing and freeze substitution were determined by transmission electron microscopy. As expected, Woronin bodies were found near septa. High numbers of morphologically identical organelles were also found in apical regions. To verify that these organelles were authentic Woronin bodies, we used antibodies raised against the Neurospora crassa Woronin body matrix protein Hex1. Anti-Hex1 antibodies labeled Woronin bodies at septa and in apical regions of A. nidulans. In germlings that had not yet formed septa, at least fifty percent of Woronin bodies were found within 2.5 μm of the tip. In germ tubes that had formed septa, the total number of Woronin bodies remained the same, but only twenty percent were near the tip. Our results clearly establish that Woronin bodies are found in apical regions of Aspergillus germ tubes and suggest that Woronin bodies are transported from the apex to the more basal regions of the cell immediately before or during septation.     

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.     

Dynamin-like protein-dependent formation of Woronin bodies in Saccharomyces cerevisiae upon heterologous expression of a single protein

Filamentous ascomycetes harbor Woronin bodies and glyoxysomes, two types of microbodies, within one cell at the same time. The dominant protein of the Neurospora crassa Woronin body, HEX1, forms a hexagonal core crystal via oligomerization and evidence has accumulated that Woronin bodies bud off from glyoxysomes. We analyzed whether HEX1 is sufficient to induce Woronin body formation upon heterologous expression in Saccharomyces cerevisiae, an organism devoid of this specialized organelle. In wild-type strain BY4742, initial import of HEX1 into existing peroxisomes enabled the formation of organelles with a hexagonal crystal. The observed structures mimicked the shape of genuine Woronin bodies, but exhibited a lower density and were significantly larger. Double-immunofluorescence analysis revealed that hexagonal HEX1 structures only occasionally co-localized with peroxisomal marker proteins, indicating that the Woronin-body-like structures are well separated from peroxisomes. In cells lacking Vps1p and Dnm1p, dynamin-like proteins required for the division of peroxisomes, the Woronin-body-like organelles remained attached to peroxisomes. The data indicate that Woronin bodies emerge after the formation of a HEX1 core crystal within peroxisomes followed by Vps1p- and Dnm1p-mediated fission.     

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.     

A HEX-1 crystal lattice required for Woronin body function in Neurospora crassa

The Woronin body is a dense-core vesicle specific to filamentous ascomycetes (Euascomycetes), where it functions to seal the septal pore in response to cellular damage. The HEX-1 protein self-assembles to form this solid core of the vesicle. Here, we solve the crystal structure of HEX-1 at 1.8 A, which provides the structural basis of its self-assembly. The structure reveals the existence of three intermolecular interfaces that promote the formation of a three-dimensional protein lattice. Consistent with these data, self-assembly is disrupted by mutations in intermolecular contact residues and expression of an assembly-defective HEX-1 mutant results in the production of aberrant Woronin bodies, which possess a soluble noncrystalline core. This mutant also fails to complement a hex-1 deletion in Neurospora crassa, demonstrating that the HEX-1 protein lattice is required for Woronin body function. Although both the sequence and the tertiary structure of HEX-1 are similar to those of eukaryotic initiation factor 5A (eIF-5A), the amino acids required for HEX-1 self-assembly and peroxisomal targeting are absent in eIF-5A. Thus, we propose that a new function has evolved following duplication of an ancestral eIF-5A gene and that this may define an important step in fungal evolution.     

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.     

The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.     

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.     

Making two organelles from one: Woronin body biogenesis by peroxisomal protein sorting

Woronin bodies (WBs) are dense-core organelles that are found exclusively in filamentous fungi and that seal the septal pore in response to wounding. These organelles consist of a membrane-bound protein matrix comprised of the HEX protein and, although they form from peroxisomes, their biogenesis is poorly understood. In Neurospora crassa, we identify Woronin sorting complex (WSC), a PMP22/MPV17-related membrane protein with dual functions in WB biogenesis. WSC localizes to large peroxisome membranes where it self-assembles into detergent-resistant oligomers that envelop HEX assemblies, producing asymmetrical nascent WBs. In a reaction requiring WSC, these structures are delivered to the cell cortex, which permits partitioning of the nascent WB and WB inheritance. Our findings suggest that WSC and HEX collaborate and control distinct aspects of WB biogenesis and that cortical association depends on WSC, which in turn depends on HEX. This dependency helps order events across the organellar membrane, permitting the peroxisome to produce a second organelle with a distinct composition and intracellular distribution.     

Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.     

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.     

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.     

Woronin bodies fromPenicillium chrysogenum: Isolation and characterization by analytical subcellular fractionation

Differential centrifugation of whole homogenates ofPenicillium chrysogenum, disrupted by a modified Ballotini bead method, resulted in the enrichment of Woronin bodies between 800g (5 minutes) and 6000g (10 minutes). Isolated Woronin bodies are membrane-bounded, electron-opaque, approximately spherical organelles, 0.11 to 0.29 μm in diameter. Woronin bodies have a buoyant density (ϱ) of 1.21 g cm⁻³ and S_20,w values of 6300 to 37,600 in sucrose gradients. Analytical subcellular fractionation of whole homogenates in a zonal rotor showed that Woronin bodies did not cosediment with marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (cytochrome c oxidase), or endoplasmic reticulum (NADPH cytochrome c reductase).     

Drosophila Signal Peptidase Complex Member Spase12 Is Required for Development and Cell Differentiation

It is estimated that half of all proteins expressed in eukaryotic cells are transferred across or into at least one cellular membrane to reach their functional location. Protein translocation into the endoplasmic reticulum (ER) is critical to the subsequent localization of secretory and transmembrane proteins. A vital component of the translocation machinery is the signal peptidase complex (SPC) - which is conserved from yeast to mammals – and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Failure to cleave the SP, due to mutations that abolish the cleavage site or reduce SPC function, leads to the accumulation of uncleaved proteins in the ER that cannot be properly localized resulting in a wide range of defects depending on the protein(s) affected. Despite the obvious importance of the SPC, in vivo studies investigating its function in a multicellular organism have not been reported. The Drosophila SPC comprises four proteins: Spase18/21, Spase22/23, Spase25 and Spase12. Spc1p, the S. cerevisiae homolog of Spase12, is not required for SPC function or viability; Drosophila spase12 null alleles, however, are embryonic lethal. The data presented herein show that spase12 LOF clones disrupt development of all tissues tested including the eye, wing, leg, and antenna. In the eye, spase12 LOF clones result in a disorganized eye, defective cell differentiation, ectopic interommatidial bristles, and variations in support cell size, shape, number, and distribution. In addition, spase12 mosaic tissue is susceptible to melanotic mass formation suggesting that spase12 LOF activates immune response pathways. Together these data demonstrate that spase12 is an essential gene in Drosophila where it functions to mediate cell differentiation and development. This work represents the first reported in vivo analysis of a SPC component in a multicellular organism.     

Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wavelength of 1.06 micron. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.     

Ss-Sl2, a novel cell wall protein with PAN modules, is essential for sclerotial development and cellular integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum.     

Vacuoles and fungal biology

Fungal vacuoles have long been recognised as versatile organelles, involved in many aspects of protein turnover, cellular homeostasis, membrane trafficking, signalling and nutrition. Recent research has also revealed an expanding repertoire of physiological functions for fungal vacuoles that are vital for fungal growth, differentiation, symbiosis and pathogenesis. Vacuole-mediated long-distance nutrient transporting systems have been shown to facilitate mycelial foraging and long-distance communication in saprophytes and mycorrhizal fungi. Some hyphae of plant and human fungal pathogens can grow under severely nutrient-limited conditions by expanding the vacuolar space rather than synthesising new cytoplasm and organelles. Autophagy has been recognised as a crucial process in plant pathogens for the initiation of appressorium formation. These studies demonstrate the importance of fungal vacuoles as organelles that are essential for many of the attributes that define the activities and roles of fungi in their natural environments.