Ultrastructural interaction between multinucleate giant cells and the fungus in aspergillomas of human paranasal sinuses

The interaction between multinucleate giant cells (MGCs) and the fungusAspergillus flavus as seen by transmission electron microscopy (TEM) is described in paranasal granulomas occurring in a Saudi patient dying from chronic aspergillosis. Two morphologically different types of MGCs were recognized; these were: a) ‘Unhealthy looking’ type I cells, rich in well organized organelles and containing few, partially degenerated and necrotic fungal elements. b) ‘Healthy looking’ type II cells that contained scanty, randomly dispersed cell organelles and normal, or partially degenerated fungal hyphae. The fungal elements had very thick and multilayered cell walls, and were found either in close contact to the host cell cytoplasm, or enclosed within phagosomes. The mechanism of the fungus destruction by the host MGCs is described and compared with that previous reports of MGCs involved in the elimination of extracellular microorganisms. The morphology and the various physiological activities of MGCs seemed to depend mainly on whether the pathogen is extra- or intracellular. However, this study showed that MGCs are the cells best suited for killing pathogenic fungi.     

Irregular growth forms and cell wall modifications,polygalacturonase detection,and endocell formation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-lycopersici</Emphasis> infecting tomato plants,as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.     

μ-chain-deficient mice possess B-1 cells and produce IgG and IgE, but Not IgA, following systemic sensitization and inhalational challenge in a fungal asthma model

Allergic bronchopulmonary aspergillosis is often difficult to treat and results in morbidity associated with chronic airway changes. This study assessed the requirement for B cells and their products in the allergic pulmonary phenotype in a murine model of fungal allergic asthma that mimics allergic bronchopulmonary aspergillosis. C57BL/6 and μMT mice (assumed to lack peripheral B cells) were sensitized with Aspergillus fumigatus extract and challenged with two inhalation exposures of live conidia to induce airway disease. Airway hyperresponsiveness after methacholine challenge, peribronchovascular inflammation, goblet cell metaplasia, and fibrotic remodeling of the airways was similar between μMT mice and their wild-type counterparts (C57BL/6). Surprisingly, even in the absence of the μ-chain, these μMT mice produced IgE and IgG Abs, although the Abs induced did not have specificity for A. fumigatus Ags. In contrast, IgA was not detected in either the lavage fluid or serum of μMT mice that had been exposed to A. fumigatus. Our findings also reveal the existence of CD19(+)CD9(+)IgD(+) B-1 cells in the lungs of the μMT animals. These data show the μMT mice to have a developmental pathway independent of the canonical μ-chain route that allows for their survival upon antigenic challenge with A. fumigatus conidia, although this pathway does not seem to allow for the normal development of Ag-specific repertoires. Additionally, this study shows that IgA is not required for either clearance or containment of A. fumigatus in the murine lung, as fungal outgrowth was not observed in the μMT animals after multiple inhalation exposures to live conidia.     

<Emphasis Type="Italic">Pityopus californicus</Emphasis>: structural characteristics of seed and seedling development in a myco-heterotrophic species

Pityopus californicus (Eastw.) H. F. Copel., a monotypic member of the Monotropoideae in the family Ericaceae, is a myco-heterotrophic species with distribution limited to the Pacific Northwest of the USA. Young embryos of P. californicus developed mycorrhizal associations in seed packets that had been buried for up to 681 days, suggesting that seeds of P. californicus may require the presence of a fungus to achieve germination. Samples of nongerminated seeds and early stages in embryo and root development were subsequently processed for light microscopy, histochemistry, and transmission electron microscopy (TEM). Nongerminated seeds possessed a thick testa, lacked a shoot and root meristem, and consisted of an embryo with large parenchymatous cells containing protein bodies and starch grains as storage reserves. In the earliest developmental stage (seed coat still attached), fungal hyphae were present on the testa surface and between the testa and embryo. This stage was followed by embryo elongation, the organization of a root apical meristem, and the development of a well-developed fungal mantle surrounding the elongated embryo. At least two morphotypes were identified based on structural characteristics of the mantle. One of these, with ascomycetous septa, had Cenococcum-like features. Late-stage embryo/early root development revealed a typical mantle and Hartig net, with fungal pegs penetrating the outer tangential walls of epidermal cells. Transfer cell-like deposits of wall material, similar to those described in Monotropa spp., enclosed fungal pegs. The development of a Hartig net and fungal pegs suggests that nutrient exchange interfaces are required for seedling development.     

Suppression of the powdery mildew pathogen by chitinase microinjected into barley coleoptile epidermal cells

An exogenous chitinase from Streptomyces griseus was introduced into coleoptile epidermal cells of barley (Hordeum vulgare) by microinjection, and the effect of injected chitinase on the growth or development of the powdery mildew pathogen (Erysiphe graminis f. sp. hordei) was examined. Prior to microinjection, an enzymatic degradation of fungal haustorium, the organ taking nutrients from host plant cells, was examined by treating fixed coleoptile epidermis harboring haustoria with this enzyme. The result showed that haustoria were effectively digested by chitinase, suggesting the effectiveness of chitinase treatment for suppressing the fungal development. Microinjection of chitinase was conducted using living coleoptile tissues inoculated with the pathogen. Epidermal cells in which the haustorial primordia had been formed, or in which the haustoria had matured, were selected as targets for injection. The result clearly indicated that injection at the stage of primordium formation was effective in completely digesting haustoria and suppressing the subsequent formation of secondary hyphae of the pathogen. In microinjection after haustorial maturation, hyphal elongation was considerably suppressed though there was no detectable morphological change in the haustoria. Thus, the present study provides the experimental basis for genetically manipulating barley to produce transgenic plants resistant to the powdery mildew disease.     

Effect of various components of the fungal cell wall on adhesion of Candida albicans to the mouth mucosa epithelium in vitro]

An influence of mannan++, its component methyl-D-mannopyranoside+ and N-acetylglucosamine on in vitro adhesion of Candida albicans strains to buccal mucosal epithelium was studied. These substances inhibited adhesion when added to adherence test in a concentration of 10 mg/ml and 25 mg/ml despite whether were added to the test incubation medium or when preincubated with fungi or epithelial cells. Preincubation of fungal cells and epithelial cells with mannan had no influence on attachment; preincubation of epithelial cells with methyl-D-mannopyranoside+ and N-acetylglucosamine decreased adherence significantly. On the other hand preincubation of fungal calls with methyl-D-mannopyranoside+ increased their adhesive properties, having no influence on adherence after preincubation of fungi with N-acetylglucosamine.     

Impact of Collimonas bacteria on community composition of soil fungi

The genus Collimonas consists of soil bacteria that have the potential to grow at the expense of living fungal hyphae. However, the consequences of this mycophagous ability for soil fungi are unknown. Here we report on the development of fungal communities after introduction of collimonads in a soil that had a low abundance of indigenous collimonads. Development of fungal communities was stimulated by addition of cellulose or by introducing plants ( Plantago lanceolata ). Community composition of total fungi in soil and rhizosphere and of arbuscular mycorrhizal fungi in roots was examined by PCR-DGGE. The introduction of collimonads altered the composition of all fungal communities studied but had no effects on fungal biomass increase, cellulose degrading activity or plant performance. The most likely explanation for these results is that differences in sensitivity of fungal species to the presence of collimonads result in competitive replacement of species. The lab and greenhouse experiments were complemented with a field experiment. Mesh bags containing sterile sand with or without collimonads were buried in an ex-arable field and a forest. The presence of collimonads had an effect on the composition of fungi invading these bags in the ex-arable site but not in the forest site.     

Transient expression of anthocyanin genes in barley epidermal cells: potential for use in evaluation of disease response genes

A transient assay is described that should allow evaluation of the role of host genes in disease response by enhancing or disrupting expression of those genes in specific cells and looking for effects on disease development. The assay also has the potential for assessing utility of host and non-host genes in enhancing resistance to disease in transgenic plants. Particle bombardment with a helium discharge particle gun was utilized to transiently express genes in epidermal cells of coleoptiles of barley (Hordeum vulgare). An anthocyanin reporter gene construct provided a means of identifying those cells that were transiently expressing introduced DNA. Optimal transient expression rates were achieved two days following bombardment with 1800 psi helium pressure, 1.0 m diameter gold particles, and coleoptile pre- and post-treatment in 0.30--0.35 m mannitol/sorbitol. Under optimal conditions, at least 35 cells expressed anthocyanin per bombardment. Transiently expressing cells were inoculated with the fungal pathogen, Erysiphe graminis f. sp. hordei, and fungal development observed. Neither the bombardment procedures, the presence of nearby dead cells, nor accumulation of anthocyanin within living cells affected fungal development in living cells. Therefore, incorporation of disease-related genes onto the same plasmid as the reporter genes will allow evaluation of the role of those genes in disease development or suppression. Since particle bombardment is possible with a great range of different plant tissues, the described methodology should exhibit wide applicability for evaluating genes in diverse plant-pathogen interactions, as well as genes involved in many other biological processes     

Subcellular distribution of marker enzymes in cells of a minute fungus, Fusidium sp. 100-3

An electron microscope cytochemical technique was used to determine the subcellular distribution of marker enzymes in Fusidium sp. 100-3 cells. Nucleoside diphosphatase was found in the nuclear envelope and intracytoplasmic membrane segment. Thiamine pyrophosphatase was found to be associated with the mesosomes. Cytochrome c (oxidase) activity was found only in the mitochondrial cristae. Strong alkaline phosphatase activity was present in the vacuole; in addition, the enzyme activity was discretely dispersed throughout the cytoplasm without any association with any membrane material. The overall characteristics of the cell ultrastructure and subcellular enzyme distribution of Fusidium sp. 100-3 cells compare fairly well with those of a fungal cell. But there are considerable differences from the characteristics of higher eucaryotic cells. Detailed data on the marker enzymes distribution in a variety of fungal cells are not available. Therefore, it is not possible to conclude whether the marker enzyme distribution of Fusidium sp. 100-3 cells is unique or is typical of any fungal organism. Detailed studies of cell ultrastructure of and marker enzyme distribution in minute fungal cells and their comparison to the ultrastructure of and marker enzyme distribution in other fungal organisms may be helpful in understanding the phylogenetic and ontogenic development of subcellular organelles.     

Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark-beetle attack in Norway spruce (Pinaceae)

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.     

Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Bark beetles have a decisive impact on fungal communities in Norway spruce stem sections

To study the importance of insects in the establishment of fungi, stem sections of Norway spruce were placed in mature managed conifer forests in Southeast Sweden. After one or two flying seasons, fungal communities in wood, bark and bark beetle samples were analysed by molecular methods. Excluding insects from stem sections with cages had a significant effect on the fungal community. Small wounds made in the bark to mimic insect activity did not significantly alter the fungal community, indicating that physical holes as such only played a minor role for the insect interaction with the fungal community development. Several white rot species were significantly more abundant in stem sections with insect access and were also detected from bark beetle samples. This suggests that insects do contribute to the development of early fungal succession on dead wood, but that creating small disturbances in the bark only have a minor contributing effect.     

Fungal S-adenosylmethionine synthetase and the control of development and secondary metabolism in Aspergillus nidulans

The filamentous fungus Aspergillus nidulans carries a single gene for the S-adenosylmethionine (SAM) synthetase SasA, whereas many other organisms possess multiple SAM synthetases. The conserved enzyme catalyzes the reaction of methionine and ATP to the ubiquitous methyl group donor SAM. SAM is the main methyl group donor for methyltransferases to modify DNA, RNA, protein, metabolites, or phospholipid target substrates. We show here that the single A. nidulans SAM synthetase encoding gene sasA is essential. Overexpression of sasA, encoding a predominantly cytoplasmic protein, led to impaired development including only small sterile fruiting bodies which are surrounded by unusually pigmented auxiliary Hülle cells. Hülle cells are the only fungal cell type which does not contain significant amounts of SasA. Sterigmatocystin production is altered when sasA is overexpressed, suggesting defects in coordination of development and secondary metabolism. SasA interacts with various metabolic proteins including methionine or mitochondrial metabolic enzymes as well as proteins involved in fungal morphogenesis. SasA interaction to histone-2B might reflect a putative epigenetic link to gene expression. Our data suggest a distinct role of SasA in coordinating fungal secondary metabolism and development.     

实验室条件下五唇兰菌根真菌专一性研究

利用从高原温带兰科植物菌根中获得的22个菌根真菌菌株, 对五唇兰(Doritis pulcherrima)进行了室内种子萌发、原球茎分化和组培苗回接试验, 从交叉回接的角度对附生兰科植物与菌根真菌的生理专一性进行了探讨。经过20周的共生培养, 只有编号为Cf1和Mm1的两个菌株使种子表现出种胚明显膨大的萌发迹象; 9个菌株能够促使原球茎较好地分化发育出根叶; 11个菌株处理苗的平均鲜重增长率高于对照组(156.25%), 其中Mm1的效果达到极显著水平(p = 0.01)。通过根切片显微观察, 在原球茎分化根和回接效果良好的处理苗的根皮层组织发现典型的菌丝团结构, 表明菌根体系已成功建立。温带地生兰菌根真菌对五唇兰种子萌发、原球茎发育和幼苗生长等3个重要生长阶段影响的试验显示, 五唇兰的种子和菌根真菌的共生萌发效果不佳, 而原球茎及幼株更容易与之建立良好的共生关系。同时, 也没有发现同一个真菌菌株能够对五唇兰的种子、原球茎和幼苗均产生促进作用。研究结果表明, 五唇兰的菌根真菌专一性因生理生长阶段的不同而存在差异。     

New insights into the infection process of Rhynchosporium secalis in barley using GFP

Through the use of a Rhynchosporium secalis isolate transformed with the green fluorescent protein gene and LASER scanning confocal microscopy (LSCM), fungal development during the R. secalis/barley interaction was analysed. Each infection stage was investigated from 0.5h to 14 days post-inoculation (p.i.) with extensive sampling within the first 48 h p.i. Early germination events were observed that had not been previously described. A specific time of germination was noted, with germ tube formation appearing as early as 1h p.i. Conidia were observed within anticlinal grooves of epidermal cells and the formation of bubbles within these pectin-rich regions was observed within 24h p.i. The study reports R. secalis pectinase production and suggests degradation of these pectin-rich regions. Reactive oxygen species were present during early penetration, 3h p.i. and co-localised with fungal development. LSCM allowed the visualisation of fungal growth deep within tissues at the later stage of the infection.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.