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Role of peg formation in clamp cell fusion of homobasidiomycete fungi.

In most filamentous basidiomycetes, clamp cells are found at the septa of dikaryotic mycelia. Clamp cell formation starts at hyphal tip cells with the development of a lateral bulge at a position slightly apical to the future septum. Relative to the growth direction of the hypha, the protrusion expands backwards into a hook-like structure. Next, the two genetically different haploid nuclei within the hyphal tip cell divide. A septum appears between clamp cell and hyphal tip cell, thereby trapping one nucleus within the clamp cell. Another septum is laid within the hypha, separating a nucleus of the other type in the newly generated subapical hyphal cell from the two different nuclei kept together in the new apical hyphal cell. Through fusion of clamp and subapical cell, the two solitary nuclei become united within the subapical hyphal compartment. In 1933, Buller described subapical formation of a peg to which the clamp cell fuses as an additional, subsequently neglected step in this series of events. In this study, we represent evidence for subapical peg formation and its role in clamp cell fusion. Our observations potentially indicate a B mating type regulated extracellular communication between clamp and subapical hyphal cell.

Agaricales↗

Two-dimensional reference map of Candida albicans hyphal forms.

We have set up an in vitro model of culture of Candida albicans fungal cells that mimics the physiological conditions found in human beings in order to carry out studies of host-pathogen interaction. Under these conditions, C. albicans undergoes dimorphic transition (yeast-hyphae); this morphological change has been proposed as a virulence factor for this fungus. C. albicans cytoplasmic extract from hyphae cells was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and a reference map was obtained. Protein identification was carried out by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or a MALDI-TOF/TOF mass spectrometer. A total of 106 spots, excised from 2-D gels, were analyzed. This resulted in the identification of 43 proteins involved in metabolism, 13 involved in transcription, protein synthesis, and fate, 8 involved in cell rescue, virulence, and defense, and 2 proteins of unknown function. This reference map is an important tool for future studies of protein differential expression after host-pathogen interaction.

Candida albicans↗

Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa.

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

Hyphae↗

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity.

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a approximately 20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.

Amino Acid Sequence↗

Saprobic potential of Tricholoma matsutake: growth over pine bark treated with surfactants.

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.

Agaricales↗

Magnesium ions alleviate the negative effect of manganese on Glomus claroideum BEG23.

The phytotoxicity of excessive levels of manganese (Mn), an essential micronutrient, can be alleviated significantly by a high supply of magnesium (Mg) ions to plants. A similar interaction of these two elements in the development of arbuscular mycorrhizal (AM) fungi has been verified in two experimental systems. In in vitro experiments, an outgrowth of hyphae from excised, surface-disinfected root segments colonised with Glomus claroideum BEG23 was measured after 5 days incubation in liquid media. When only Mn ions were present in the media at higher concentrations (>0.05 mM), the growth of hyphae from root segments was reduced significantly. Addition of magnesium sulphate to the incubation solution reduced the inhibitory effects of Mn on hyphal growth. Alleviation of Mn toxicity by Mg ions observed in in vitro experiments was verified also for the symbiotic association between G. claroideum and maize as a host plant in a hydroponics sand culture experiment.

Culture Media↗

Apical growth and mitosis are independent processes in Aspergillus nidulans.

It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.

Aspergillus nidulans↗

Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks.

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.

Animals↗

Fungal degradation of calcium-, lead- and silicon-bearing minerals.

The aim of this study was to examine nutritional influence on the ability of selected filamentous fungi to mediate biogenic weathering of the minerals, apatite, galena and obsidian in order to provide further understanding of the roles of fungi as biogeochemical agents, particularly in relation to the cycling of metals and associated elements found in minerals. The impact of three organic acid producing fungi (Aspergillus niger, Serpula himantioides and Trametes versicolor) on apatite, galena and obsidian was examined in the absence and presence of a carbon and energy source (glucose). Manifestation of fungal weathering included corrosion of mineral surfaces, modification of the mineral substrate through transformation into secondary minerals (i.e. crystal formation) and hyphal penetration of the mineral substrate. Physicochemical interactions of fungal metabolites, e.g. H+ and organic acids, with the minerals are thought to be the primary driving forces responsible. All experimental fungi were capable of mineral surface colonization in the absence and presence of glucose but corrosion of the mineral surface and secondary mineral formation were affected by glucose availability. Only S. himantioides and T. versicolor were able to corrode apatite in the absence of glucose but none of the fungi were capable of doing so with the other minerals. In addition, crystal formation with galena was entirely dependent on the availability of glucose. Penetration of the mineral substrates by fungal hyphae occurred but this did not follow any particular pattern. Although the presence of glucose in the media appeared to influence positively the mineral penetrating abilities of the fungi, the results obtained also showed that some geochemical change(s) might occur under nutrient-limited conditions. It was, however, unclear whether the hyphae actively penetrated the minerals or were growing into pre-existing pores or cracks.

Aspergillus niger↗

Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans.

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 microg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4-2.0 microg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 microg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 microg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.

Gas Chromatography-Mass Spectrometry↗

Candida albicans protein analysis during hyphal differentiation using an integrative HA-tagging method.

In order to understand the biological complexity inherent to the pathogenicity of Candida albicans, gene expressions need to be analyzed at the protein level. We employed an epitope-tagging technique in a set of C. albicans ORF, and constructed fifteen strains which expressed HA-tagged proteins. These efforts permitted us to identify differentially synthesized proteins during the hyphal differentiations. ICL1, MLS1, and WAP1, all of which are known to be hypha-induced at the transcript level, were indeed found to be up-regulated at the protein level. We also identified CaeIF4G, CaTPO5, and CaZRT1, the protein levels of which were increased during hyphal transition, and CaERB1, the protein level of which was reduced consistently. The hypha-induced protein level of CaeIF4G was closely associated with the cellular hyphal phenotype. CaeIF4G overexpression was shown to result in hyperfilamentation in C. albicans. CaeIF4E, which was constitutively expressed during the hyphal development, exhibited no overexpression phenotype. HA-tagged strains were also utilized in our analysis of C. albicans proteins in a co-culture of macrophage and C. albicans. Five genes were found to be expressed differentially during the macrophage co-cultures. Our approaches proved to be rather useful under yeast culture conditions as well as in co-cultures of macrophage and C. albicans.

Candida albicans↗

A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation.

Upon germination Aspergillus nidulans swoM1 exhibits abnormal development by extending a primary germ tube that quickly reverts to isotropic growth and results in an enlarged, swollen apex with pronounced wall thickenings. Apical lysis occurs in 38% of the germlings. A point mutation in the AN6037.3 gene encoding the only phosphoglucose isomerase in A. nidulans is responsible for the defect. Loss of polarity is bypassed when glucose is replaced with alternate carbon sources but in all cases the mutant is unable to conidiate due to a block in conidiophore development at vesicle formation. In conidiophores SwoM::GFP localizes to multiple punctate, foci within each actively growing cell type, and to multiple foci in mature dormant conidia. In hyphae SwoM::GFP localized to two rings spanning the center of mature septa. In hyphae localization is concentrated at actively growing hyphal tips.

Aspergillus nidulans↗

Morphogenesis and cell cycle progression in Candida albicans.

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenkörper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.

Candida albicans↗

Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection.

Few human pathogens possess the ability exhibited by Candida albicans to colonize and cause symptomatic infections at different body sites. The host immune system is the major factor determining whether this opportunistic yeast behaves as a commensal or as a pathogen, since C. albicans strains appear capable of expressing similar virulence factors in response to specific body-district cues. This report provides evidence showing that C. albicans isolates with diverse genomic backgrounds (b and c karyotypes) differently modulate their pathogenic potential when assayed in cocultures with human monocytic derived macrophages (THP-1 cells). Striking differences were observed in the ability to undergo bud-hypha transition, a relevant C. albicans virulence factor, between b and c karyotypes (P<0.0001) upon their internalization by macrophages. All c types were able to develop hyphal forms, resist intracellular killing, replicate, and escape from macrophages. The b type isolates, which were shown to be more efficiently ingested by THP-1 cells than the c type strains (P=0.013), were susceptible to intracellular killing and predominantly found as blastoconidia inside macrophages. Despite their different intracellular disposition, both b and c type isolates were equally able to undergo morphogenesis and to express NRG1 and HWP1 genes, markers of the bud-hypha transition program, during in vitro propagation. Since macrophages play a critical role in the host resistance to C. albicans, the different response of b and c isolates to macrophage infection suggests that the c type strains are better suited to behave as a more virulent strain cluster.

Candida albicans↗

Hassiella monospora gen. et sp. nov., a microfungus from the 400 million year old Rhynie chert.

A new microfungus, Hassiella monospora gen. et sp. nov., consisting of coenocytic hyphae is associated with degraded plant material in the Early Devonian silicified Rhynie chert ecosystem. Some hyphae produce small bulb-like projections that subsequently develop into spherical, thick-walled and highly ornamented reproductive structures. Mature reproductive structures are characterized by a prominent, funnel-shaped appendage that is interpreted as an amphigynous antheridium. When combined, these features are suggestive of the oogonia/oosporangia in certain extant members of the Peronosporomycetes (Oomycota).

Ecosystem↗

Fungal surface remodelling visualized by atomic force microscopy.

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 degrees and 42 degrees C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 degrees C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 degrees C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 degrees C) to permissive (28 degrees C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.

Aspergillus nidulans↗

Adaptation of the Efg1p morphogenetic pathway in Candida albicans by negative autoregulation and PKA-dependent repression of the EFG1 gene.

The Efg1p regulator protein permits hyphal morphogenesis in the human fungal pathogen Candida albicans. We have identified the major promoter of the EFG1 gene as a direct target of Efg1p, resulting in negative autoregulation of EFG1. Enhanced activity of protein kinase A (PKA) isoforms Tpk1p and Tpk2p or exogenous overexpression of EFG1 led to Efg1p-dependent down-regulation of the endogenous EFG1 promoter. Serial deletion analyses of the promoter region revealed that the TATA box region was required for EFG1 autoregulation. By chromatin immunoprecipitation we detected binding of Efg1p to the EFG1 transcriptional initiation region. Furthermore, Sin3p, a component of a specific histone deacetylase complex, was shown to bind to the EFG1 promoter. sin3 mutants grew as budding pseudohyphae and were unable to form true hyphae, similar to strains constitutively expressing EFG1. We propose that the PKA signalling pathway, in addition to its importance in the initial steps of filament formation, is part of a feedback loop that controls EFG1 expression allowing continued hypha formation in inducing conditions. This autoregulation of EFG1 expression is probably mediated through the Sin3p-containing histone deacetylation complex.

Adaptation, Physiological↗

The role of arbuscular mycorrhiza in zinc uptake by red clover growing in a calcareous soil spiked with various quantities of zinc.

Three pot experiments were conducted to investigate the role of arbuscular mycorrhiza (AM) in Zn uptake by red clover. Plants inoculated with Glomus mosseae and uninoculated controls were grown in a sterile calcareous soil in 'Plexiglas' (Acrylic) containers with two nylon net partitions (30 microm mesh) to separate the central root zone from the two outer hyphal zones. The effects of mycorrhiza on plant growth and Zn uptake changed dramatically with increasing Zn addition level (range 0-1200 mg kg(-1)) in the root zone. With Zn addition levels <300 mg kg(-1), added Zn did not affect plant yield and above the critical level plant yield gradually decreased but was always higher for mycorrhizal than for controls. Below the critical Zn application rate (50 mg kg(-1)), Zn uptake was enhanced while above this level Zn translocation to the shoots decreased. At all Zn addition levels, mycorrhizal colonization increased Zn absorption and accumulation in the roots, and this may help to explain the alleviation of Zn toxicity at high Zn application rates. As expected, AM colonization enhanced P nutrition and hence yield at all added Zn levels studied. Efforts were made to obtain direct evidence for hyphal contribution to Zn uptake by applying both Zn to the hyphal growth zone and additional P to the root zone to avoid the 'growth dilution effect'. The data demonstrate that mycorrhizal hyphae could absorb Zn directly from the soil and then transfer it to the plant roots. The hyphal contribution to Zn uptake by the host plant reached its maximum value at the Zn addition level of 50 mg kg(-1), in which Zn uptake via the extramatrical hyphae comprised 22% of total uptake, thus confirming the critical Zn application level found previously.

Hyphae↗