High spatial resolution analysis of fungal cell biochemistry--bridging the analytical gap using synchrotron FTIR spectromicroscopy.

Fungi impact humans and the environment in many ways, for good and ill. Some fungi support the growth of terrestrial plants or are used in biotechnology, and yet others are established or emerging pathogens. In some cases, the same organism may play different roles depending on the context or the circumstance. A better understanding of the relationship between fungal biochemical composition as related to the fungal growth environment is essential if we are to support or control their activities. Synchrotron FTIR (sFTIR) spectromicroscopy of fungal hyphae is a major new tool for exploring cell composition at a high spatial resolution. Brilliant synchrotron light is essential for this analysis due to the small size of fungal hyphae. sFTIR biochemical characterization of subcellular variation in hyphal composition will allow detailed exploration of fungal responses to experimental treatments and to environmental factors.     

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

森林生态系统中木腐真菌群落形成机理及生态功能

在森林生态系统中,枯死木是一个重要的组成部分,为很多生物提供栖息地,有助于养分循环以及碳和水的储存.木材分解是森林生态系统养分循环、土壤形成和碳收支的决定性过程,越来越受到森林生态学家、病理学家和管理者的重视.在此过程中,木腐真菌通过分泌多种酶降解木材主要成分,实现生态系统中的物质循环,具有极为关键和重要的作用.木腐真...     

Relationship between fluorescein diacetate-stained hyphae and oxygen utilization, glucose utilization, and biomass of submerged fungal batch cultures.

The relationship between fungal activity and staining with fluorescein diacetate (FDA) was investigated by growing Penicillium citrinum and Rhizoctonia solani in submerged batch cultures at different initial glucose concentrations and aeration rates. A modified FDA staining method, similar to the Jones and Mollison technique (P. Jones and J. Mollison, J. Gen. Microbiol. 2:54-69, 1948), was developed to assess both total and FDA-stained hyphae. In previous studies, soil hyphae stained with FDA were considered viable. However, determination of a quantitative relationship between FDA staining and fungal activity is necessary before such an assumption can be made. Growth rates and the rate of change in the percentage of FDA-stained hyphae were significantly correlated. The regression equation calculated for the relationship was: growth rate (mg . ml-1 . h-1) = 0.34 + 1.1 (rate of change in the percentage of FDA-stained hyphae [. ml-1 . h-1]). Changes in activity as measured by O2 utilization, glucose utilization, and biomass correlated significantly with changes in the percentage of FDA-stained hyphae, although the relationships among these parameters were different for each fungal species. Fungal growth stage was also correlated with the percentage of FDA-stained hyphae. Staining was 10% or greater during fungal growth and less than 10% during the late growth, stationary, and death phases. Thus, the rate of change in the percentage of FDA-stained hyphae can be used to predict fungal activity rate changes for single fungal cultures and growth rates for mixed fungal cultures, and the growth stage can be assessed by the percentage of FDA-stained hyphae.     

Laser microdissection of fungal septa as visualised by scanning electron microscopy

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.     

Growing edible mushrooms: a conversation between bacteria and fungi

Mushroom cropping consists of the development and fructification of different fungal species in soil or selective substrates that provide nutrients and support for the crop. The microorganisms present in these environments strongly influence, and in some cases are required for the growth and fructification of cultivated mushrooms. Some fungi such as truffles and morels form ectomycorrhizal associations with host plants. For these fungi, helper bacteria play an important role in the establishment of plant-fungal symbioses. Selective processes acting on the microbiota present in substrates and soils determine the composition of the microbiota inhabiting the fruit bodies or interacting with fungal hyphae, and both configure the mushroom holobiont, understood as the fungus plus associated microorganisms. Here, we review current knowledge regarding the cross-talk between bacteria and fungi during mushroom cultivation. We highlight the potential use of bioinoculants as agronomical amendments to increase mushroom productivity through growth promotion or as biocontrol agents to control pests and diseases.     

A Fungal-Prokaryotic Consortium at the Basalt-Zeolite Interface in Subseafloor Igneous Crust

We have after half a century of coordinated scientific drilling gained insight into Earth´s largest microbial habitat, the subseafloor igneous crust, but still lack substantial understanding regarding its abundance, diversity and ecology. Here we describe a fossilized microbial consortium of prokaryotes and fungi at the basalt-zeolite interface of fractured subseafloor basalts from a depth of 240 m below seafloor (mbsf). The microbial consortium and its relationship with the surrounding physical environment are revealed by synchrotron-based X-ray tomographic microscopy (SRXTM), environmental scanning electron microscopy (ESEM), and Raman spectroscopy. The base of the consortium is represented by microstromatolites—remains of bacterial communities that oxidized reduced iron directly from the basalt. The microstromatolites and the surrounding basalt were overlaid by fungal cells and hyphae. The consortium was overgrown by hydrothermally formed zeolites but remained alive and active during this event. After its formation, fungal hyphae bored in the zeolite, producing millimetre-long tunnels through the mineral substrate. The dissolution could either serve to extract metals like Ca, Na and K essential for fungal growth and metabolism, or be a response to environmental stress owing to the mineral overgrowth. Our results show how microbial life may be maintained in a nutrient-poor and extreme environment by close ecological interplay and reveal an effective strategy for nutrient extraction from minerals. The prokaryotic portion of the consortium served as a carbon source for the eukaryotic portion. Such an approach may be a prerequisite for prokaryotic-eukaryotic colonisation of, and persistence in, subseafloor igneous crust.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.     

烟曲霉细胞壁的组分、组装及其功能概述

环境中普遍存在的腐生丝状真菌烟曲霉Aspergillus fumigatus在免疫功能低下或缺陷的人群中可引起多种急慢性疾病,包括致死率很高的侵袭性曲霉病。细胞壁作为真菌的细胞外骨架结构不仅起维持细胞形状、保护细胞抵抗外界压力等作用,在病原真菌极性生长、入侵新的生态域、启动宿主免疫反应中也起重要作用。细胞壁组分还是真菌感染的分子诊断基础和开发抗真菌药物的理想靶标。近几十年来烟曲霉细胞壁的遗传、生物化学及免疫学方向的研究使其成为研究真菌细胞壁的模式真菌。本文主要概述烟曲霉细胞壁的组分、分子组装机制及其在真菌生存和感染中的作用,并对未来研究方向提出了展望。     

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans

Candida albicans , the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 °C under aerobic conditions with 5% CO₂. Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.     

Monoclonal antibodies incorporated into Neotyphodium coenophialum fungal cultures: inhibition of fungal growth and stability of antibodies.

Three monoclonal antibodies (mAbs) produced against proteins from the tall fescue (Festuca arundinacea Schreb.) fungal endophyte Neotyphodium coenophialum hybridize exclusively to a fungal protein under denaturing conditions. The protein is approximately 88 kDa in size. These mAbs were individually incorporated into liquid medium to determine their effects on fungal growth in culture. Neotyphodium-specific mAbs inhibited fungal growth for the duration of the study. Fungal cultures grown in the presence of Neotyphodium-naive mAbs or in the absence of all mAbs grew unimpeded. Bright-field microscopy and immunohistochemical studies of cultures containing Neotyphodium-specific mAbs revealed a change in mycelia morphology with clumps exhibiting a gelatinous matrix containing sparse hyphae, while cultures receiving Neotyphodium-naive mAbs in medium demonstrated unrestricted growth with overlapping and branched hyphae. In liquid culture devoid of fungal isolates, mAbs were stable and detected throughout the experiment, but were below threshold detection levels within 15 min following inclusion in liquid cultures containing Neotyphodium spp., indicating rapid binding to fungal mycelia. Monoclonal antibodies may provide a new method to help control plant pathogenic fungi where chemical or genetic means are not feasible.     

Antifungal effects of essential oils from oregano and fennel on Sclerotinia sclerotiorum

AIMS: The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotiorum. Effects of the essential oils on morphological structures of hyphae and sclerotia were studied under light and scanning electron microscopes (SEM). METHODS AND RESULTS: Inhibitory effects of volatile and contact phases of the essential oils used were determined on hyphae and sclerotia. Both essential oils have a marked antifungal effect against S. sclerotiorum. Soil amendment with essential oils has significant effect on reducing sclerotial viability. Both essential oils significantly inhibited the fungal growth in soil, thereby increasing the number of surviving tomato seedling by 69.8% and 53.3%, respectively. Light and SEM observations on pathogen hyphae and sclerotia revealed considerable morphological alterations in hyphae and sclerotia. CONCLUSIONS: The significant reduction in the mycelial growth and germination of sclerotia would greatly reduce the pathogen inoculum source. This may influence the rate of disease development in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the reduction in the number of diseased plants in infested soil amended with essential oils, we concluded that oregano and fennel essential oils could be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi.     

Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate. However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils. An unidentified group of beta-subclass Proteobacteria (CbetaPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils. Here we demonstrate that the CbetaPs grew at the expense of extending fungal mycelium of three dune soil fungi (Chaetomium globosum, Fusarium culmorum, and Mucor hiemalis) under nutrient-limiting, soil-like conditions. Aggregates of CbetaPs were also often found attached to fungal hyphae. The growth of a control group of dominant nonchitinolytic dune soil bacteria (beta- and gamma-subclass Proteobacteria) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae. Therefore, mycolytic activities of CbetaPs have apparently been involved in allowing them to grow after exposure to living hyphae. The chitinase inhibitor allosamidin did not, in the case of Mucor, or only partially, in the cases of Chaetomium and Fusarium, repress mycolytic growth of the CbetaPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation. Chitinolytic Stenotrophomonas-like and Cytophaga-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.     

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura-blaster protocol, then placing the remaining allele under the control of the maltose-inducible, glucose-repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Deltachs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon-like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.     

Nutrient uptake in mycorrhizal symbiosis

The role of mycorrhizal fungi in acquisition of mineral nutrients by host plants is examined for three groups of mycorrhizas. These are; the ectomycorrhizas (ECM), the ericoid mycorrhizas (EM), and the vesicular-arbuscular mycorrhizas (VAM). Mycorrhizal infection may affect the mineral nutrition of the host plant directly by enhancing plant growth through nutrient acquisition by the fungus, or indirectly by modifying transpiration rates and the composition of rhizosphere microflora. A capacity for the external hyphae to take up and deliver nutrients to the plant has been demonstrated for the following nutrients and mycorrhizas; P (VAM, EM, ECM), NH₄ ⁺ (VAM, EM, ECM), NO₃ ^- (ECM), K (VAM, ECM), Ca (VAM, EM), SO₄ ^2- (VAM), Cu (VAM), Zn (VAM) and Fe (EM). In experimental chambers, the external hyphae of VAM can deliver up to 80% of plant P, 25% of plant N, 10% of plant K, 25% of plant Zn and 60% of plant Cu. Knowledge of the role of mycorrhiza in the uptake of nutrients other than P and N is limited because definitive studies are few, especially for the ECM. Although further quantification is required, it is feasible that the external hyphae may provide a significant delivery system for N, K, Cu and Zn in addition to P in many soils. Proposals that ECM and VAM fungi contribute substantially to the Mg, B and Fe nutrition of the host plant have not been substantiated. ECM and EM fungi produce ectoenzymes which provide host plants with the potential to access organic N and P forms that are normally unavailable to VAM fungi or to non mycorrhizal roots. The relative contribution of these nutrient sources requires quantification in the field. Further basic research, including the quantification of nutrient uptake and transport by fungal hyphae in soil and regulation at the fungal-plant interface, is essential to support the selection and utilization of mycorrhizal fungi on a commercial scale.