The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.     

Arbuscular mycorrhizal fungi reveal distinct patterns of anastomosis formation and hyphal healing mechanisms between different phylogenic groups

The significance of anastomosis formation and the hyphal healing mechanism (HHM) for functionality and integrity of the arbuscular mycorrhizal (AM) fungal mycelial network remains poorly documented. Four Glomeraceae and three Gigasporaceae were cultured monoxenically. Anastomosis formation was assessed using the grid line method, while HHM was time-lapse monitored. In intact mycelial networks, the number of anastomosis per hyphal length was higher for Glomeraceae than for Gigasporaceae strains. Glomeraceae strains studied always formed anastomosis between different hyphae, whereas anastomosis in the Gigasporaceae more often concerned hyphal bridges within the same hyphae. In both families the HHM corresponded to a four-step process; first septum formation; second initiation of growing hyphal tips (GHTs); third GHT elongation, orientation and contact; and fourth GHT fusion and cytoplasmic/protoplasmic flux re-establishment. These four steps differentiated Glomeraceae from Gigasporaceae. The type and number of anastomosis per hyphal length, and the HHM differed considerably between Glomeraceae and Gigasporaceae families representing a supplementary character that distinguishes these two families and may be of significance in ecological studies of AM fungi.     

Anastomosis formation and nuclear and protoplasmic exchange in arbuscular mycorrhizal fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 +/- 0.06 microm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus

Rice blast disease is caused by the hemibiotrophic fungus Magnaporthe oryzae, which invades living plant cells using intracellular invasive hyphae (IH) that grow from one cell to the next. The cellular and molecular processes by which this occurs are not understood. We applied live-cell imaging to characterize the spatial and temporal development of IH and plant responses inside successively invaded rice (Oryza sativa) cells. Loading experiments with the endocytotic tracker FM4-64 showed dynamic plant membranes around IH. IH were sealed in a plant membrane, termed the extra-invasive hyphal membrane (EIHM), which showed multiple connections to peripheral rice cell membranes. The IH switched between pseudohyphal and filamentous growth. Successive cell invasions were biotrophic, although each invaded cell appeared to have lost viability when the fungus moved into adjacent cells. EIHM formed distinct membrane caps at the tips of IH that initially grew in neighboring cells. Time-lapse imaging showed IH scanning plant cell walls before crossing, and transmission electron microscopy showed IH preferentially contacting or crossing cell walls at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Analysis of biotrophic blast invasion will significantly contribute to our understanding of normal plant processes and allow the characterization of secreted fungal effectors that affect these processes.     

细脚拟青霉不同菌株间的菌丝联结现象观察及其毒力测定

用 Giemsa 染色观察了细脚拟青霉(Paecilomyces tenuipes)803和1401菌株间的菌丝联结现象,观察了菌丝、孢子的细胞核数目。孢子均是单核,菌丝细胞有1—2个核,多数为单核,菌丝尖端有1—6个核。毒力测定结果表明:能形成异核体的亲和单孢子菌株组合803-17和1401-9、803-3和1401-8的混合培养物毒力与未经培养的相同菌株组合的菌丝体混合物毒力显著不同。在讨论申提出这种差异的原因是:在 Sm 液体培养中,混合接种803-17和1401-9、803-3和1410-8,经11天的混合培养后,混合培养物中形成了与核体亲本菌丝(803-17、1401-9、803-3、1401-8)毒力显著不同的异核菌丝体(803-17×1401-9)、(803-3×1401-8)。     

The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa

The process of cell fusion is a basic developmental feature found in most eukaryotic organisms. In filamentous fungi, cell fusion events play an important role during both vegetative growth and sexual reproduction. We employ the model organism Neurospora crassa to dissect the mechanisms of cell fusion and cell-cell communication involved in fusion processes. In this study, we characterized a mutant with a mutation in the gene so, which exhibits defects in cell fusion. The so mutant has a pleiotropic phenotype, including shortened aerial hyphae, an altered conidiation pattern, and female sterility. Using light microscopy and heterokaryon tests, the so mutant was shown to possess defects in germling and hyphal fusion. Although so produces conidial anastomosis tubes, so germlings did not home toward wild-type germlings nor were wild-type germlings attracted to so germlings. We employed a trichogyne attraction and fusion assay to determine whether the female sterility of the so mutant is caused by impaired communication or fusion failure between mating partners. so showed no defects in attraction or fusion between mating partners, indicating that so is specific for vegetative hyphal fusion and/or associated communication events. The so gene encodes a protein of unknown function, but which contains a WW domain; WW domains are predicted to be involved in protein-protein interactions. Database searches showed that so was conserved in the genomes of filamentous ascomycete fungi but was absent in ascomycete yeast and basidiomycete species.     

Cytological analysis of mycelial incompatibility in Rosellinia necatrix

When the mycelia of Rosellinia necatrix encounter mycelia with a different genetic background, distinct barrage lines form. In this study, we observed hyphal interactions between compatible and incompatible R. necatrix pairs by means of light and electron microscopy. Although we observed perfect hyphal anastomosis in compatible pairs of isolates, the hyphae never anastomosed in incompatible pairs (i.e., the hyphae remained parallel or crossed over without merging). These behaviours appeared to result from the detection of or failure to detect one or more diffusible factors. The attraction to other hyphae in pairs of incompatible isolates was increased by supplementation of the growing medium with activated charcoal, although no anastomosis was observed and ultrastructural observation confirmed a lack of hyphal anastomosis. Programmed cell death (PCD) started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA fragmentation were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma and nuclear membranes, suggesting that the PCD observed in heterogenic incompatibility of R. necatrix was a vacuole-mediated process.     

Exocytosis and growth do not occur only at hyphal tips

Mycologists have put extreme emphasis on hyphal tip growth as the primary mode of growth in filamentous fungi. Much attention has also been focused on the exocytosis of extracellular enzymes from hyphal tips. However, growth and exocytosis commonly occur at hyphal locations other than tips. Here I briefly review our limited understanding of growth and exocytosis during intercalary hyphal extension, subapical branch initiation, septum formation and secondary wall thickening. Secretion of extracellular enzymes and adhesion molecules from subapical hyphal regions is also discussed. Recent research using advanced live-cell imaging techniques (e.g. Hayakawa et al., 2011 in this issue) is providing new insights into the mechanistic basis of many of these processes.     

Use of DAPI for Anastomosis Group Typing of Strains of the Fungus Rhizoctonia solani

Strains of Rhizoctonia solani, a common soil-borne, pathogenic fungus of plants, are assigned to one of 11 anastomosis groups (AGs) based on the occurrence of imperfect fusions (anastomoses) between hyphae of a non-typed strain and a tester strain of one of the 11 AG's. Imperfect fusion is characterized by the death of one or more cells in each of the hyphae involved in the fusion. Although hyphae from branches of the same strain of JR. solani may fuse with each other (self-fusion), cell death does not occur. Cell death is accompanied by nuclear degradation and granulation, or plasmolysis of the cytoplasm, which often is not visible using bright-field microscopy. When the DNA-binding fluorochrome DAPI (4', 6-diamidino-2-phenylindole) is used and the hyphal fusions viewed under fluorescence microscopy, no nuclei are observed in fused hyphal cells from two strains of the same AG of R. solani Because DAPI reacts only with living nuclei, lack of staining is presumptive evidence that the fused cells are dead as a result of imperfect fusion. The use of DAPI reduces the time required for making AG determinations compared to standard methods because it eliminates the need to assess cell wall dissolution and cytoplasmic fusion. Also, it is not necessary to trace the hyphae involved in the fusion to their respective origins to ensure that self-fusion has not occurred.     

Anastomosis Formation and Nuclear and Protoplasmic Exchange in Arbuscular Mycorrhizal Fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 ± 0.06 μm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

Origin and ultrastructure of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum

A cell observation chamber was designed to perform continuous photomicroscopic observations of hyphal anastomosis and the origin of intra-hyphal hyphae in Trichophyton terrestre and T. rubrum. These data were correlated with ultrastructural features of intra-hyphal hyphae. Hyphal fusions occurred commonly in either species of Trichophyton when incubated alone. In T. terrestre, empty phyphal segments adjoined by live units were invaded at the septa from both directions by new hyphal ingrowth. Continuous observations revealed that the intra-hyphal hyphae subsequently anastomosed via a lateral fusion peg. Similar intra-hyphal hyphae were shown in T. rubrum. Electron microscopic studies revealed ascomycetous septa in both conventional hyphae and intra-hyphal hyphae. For the latter, the cytoplasm and wall of the inner hypha were bounded by cytoplasmic organelles and another cell wall of the outer hypha.     

Optical tweezer micromanipulation of filamentous fungi

Optical tweezers have been little used in experimental studies on filamentous fungi. We have built a simple, compact, easy-to-use, safe and robust optical tweezer system that can be used with brightfield, phase contrast, differential interference contrast and fluorescence optics on a standard research grade light microscope. We have used this optical tweezer system in a range of cell biology applications to trap and micromanipulate whole fungal cells, organelles within cells, and beads. We have demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; make piconewton force measurements; mechanically stimulate hyphal tips; and deliver chemicals to localized regions of hyphae. Significant novel experimental findings from our study were that germ tubes generated significantly smaller growth forces than leading hyphae, and that both hyphal types exhibited growth responses to mechanical stimulation with optically trapped polystyrene beads. Germinated spores that had been optically trapped for 25 min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Aspergillus fumigatus RasA regulates asexual development and cell wall integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.     

Response ofZea mays roots to infection withPhytophthora cinnamomi

Summary Zea mays is a non-host ofPhytophthora cinnamomi; plants survive contact with this fungus both in the field and in pot trials. TheZ. mays-P. cinnamomi interaction has been studied by light and electron microscopy. In the epidermal layer, fungal hyphae grow intercellularly through the middle lamella. This is always the case for the first hyphal contact with any cell. Hyphae making second or subsequent contacts with a cell grow preferentially between the cell wall and plasma membrane of the infected cell rather than through the middle lamella.Papillae (callose deposits) are formed in response to some, but not all, regions of contact between the plant cell and the hypha. They do not completely encase the hypha and do not stop hyphal growth. The plasma membrane-cell wall interface of the host cell must be intact for effective papilla formation, as papillae are rarely formed when the hyphae grow between the plasma membrane and the cell wall.     

Apical assemblies of intermediate filament‐like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae

Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled‐coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament‐like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP‐mCherry fusion protein, we show that FilP accumulates in gradient‐like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Action and inertia in the study of hyphal growth

Hyphae are microscopic filaments that elongate and branch to create networks of interconnected tubes. Understanding how they work remains a formidable challenge in experimental mycology. Important advances in hyphal research in the 20^th century came from electron microscopy, which revealed clusters of cytoplasmic vesicles in the cell apex, and biochemical studies that identified the cell wall materials that are assembled at the tip. Early genetic experiments on hyphae based on mutant analysis were disappointing and provided little information on the relationship between genotype and phenotype. Progress has come more recently, in the first decades of this century, by combining the techniques of molecular genetics with modern imaging methods. Live-cell imaging has allowed us to study the dynamics of cell components in strains of fungi engineered with plasmids encoding proteins fused to fluorescent probes. This technology has provided significant insights on the growth process and yet the fundamentals of hyphal growth remain elusive.