丛枝菌根真菌脂类代谢对共生信号调控的响应和反馈

丛枝菌根(AM)真菌是自然生态系统中分布最为广泛的真菌之一,在自然界物质循环和能量流动中发挥着重要作用。经过长期的协同进化,AM真菌和宿主植物之间形成了完美的互惠互利的共生关系,而真菌的脂类代谢可能是揭示共生秘密的关键所在。本文综述了AM真菌脂类代谢在共生关系建立和维持中关键作用的最新研究进展,重点探讨了AM真菌脂类代谢对共生信号调控的响应和反馈机制,主要包括:AM真菌脂类存储和释放对共生和非共生状态的响应,以及脂类代谢产物变化与共生营养传递之间的关系;脂类分解过程在共生建立初期对信号分子调控发生的响应,以及相应的物质转化和能量代谢;菌根共生互惠互利关系维持中,真菌脂类代谢与信号分子交流通道的相互渗透和影响。本文对于理解菌根共生机制,促进菌根在生产中的应用具有促进作用。     

Epicuticular hydrocarbons of the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

The epicuticular hydrocarbons of the larval, pupal and adult stages of the sugarcane borer Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) are analysed. Dramatic changes are observed between the stages studied. Adult hydrocarbons are mostly saturated, with a predominance of 1–4 methyl‐branched straight carbon skeletons of 37–47 atoms; the major components are isomeric mixtures of internally branched trimethylderivatives of C39, C37 and C41 carbon backbones. By contrast, very small amounts of methyl‐branched components are detected in the pupae, although straight chain hydrocarbons of 23–35 carbons are the prevailing structures (70.7 ± 3.4%) with n‐C29 and n‐C27 as the major components. Unsaturated hydrocarbons (29.0 ± 3.5%) of similar chain lengths elute by gas chromatography of epicuticular extracts as complex mixtures of mono‐, di‐ and trienes; with the degree of unsaturation increasing with chain length. This is the first report of very long chain unsaturated hydrocarbons in cuticular extracts of a larval lepidopteran (93.3 ± 0.6% of the lipid components), with chain lengths in the range 37–53 carbons and up to four double bonds; the major component being C49:3, which co‐elutes with C49:4 and C49:2.     

Integrity under stress: Host membrane remodelling and damage by fungal pathogens

Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore‐forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent “stretching” of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.     

Rapid and sensitive bioassay to study signals between root exudates and arbuscular mycorrhizal fungi**

A sensitive bioassay was developed to provide a way to detect chemical signals from host plants which induce changes in hyphal growth patterns of germinated spores of arbuscular mycorrhizal (AM) fungi. The assay can be used to test host root exudates, as well as particulate fractions (root cap border cells and root mucilage), for their ability to affect AM fungal growth. Hyphal branching, induced by various host root components, can be detected as early as 4 h although results of the bioassay were usually determined after 16 to 24 h. The type of branching pattern observed was dose-dependent.     

Innate and adaptive determinants of host susceptibility to medically important fungi

The host response is the outcome of an interplay between innate immunity, adaptive immunity (Th1, Th2, T regulatory cells, B cells and antibodies) and fungal virulence factors. Dendritic cells are the gatekeepers between innate and adaptive immunity and have been the intense focus of recent studies on innate immunity to fungi because of their ability to distinguish between different forms of a fungal species, to drive Th1 versus Th2 versus T regulatory responses, and potentially be modulated by fungal products. New mechanisms have been described by which anti-fungal antibodies can modulate infection and augment T cell immunity. Th1 responses are central to limiting infection with many fungi; thus, a great deal of attention has been focused recently on the antigen(s) that trigger such a response.     

Lipid biology in fungal stress and virulence: Entomopathogenic fungi

Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between the insect host and entomogenous fungi.     

The role of the cell wall in fungal pathogenesis

Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.     

Mycorrhizal fungal growth responds to soil characteristics,but not host plant identity,during a primary lacustrine dune succession

Soil factors and host plant identity can both affect the growth and functioning of mycorrhizal fungi. Both components change during primary succession, but it is unknown if their relative importance to mycorrhizas also changes. This research tested how soil type and host plant differences among primary successional stages determine the growth and plant effects of arbuscular mycorrhizal (AM) fungal communities. Mycorrhizal fungal community, plant identity, and soil conditions were manipulated among three stages of a lacustrine sand dune successional series in a fully factorial greenhouse experiment. Late succession AM fungi produced more arbuscules and soil hyphae when grown in late succession soils, although the community was from the same narrow phylogenetic group as those in intermediate succession. AM fungal growth did not differ between host species, and plant growth was similarly unaffected by different AM fungal communities. These results indicate that though ecological filtering and/or adaptation of AM fungi occurs during this primary dune succession, it more strongly reflects matching between fungi and soils, rather than interactions between fungi and host plants. Thus, AM fungal performance during this succession may not depend directly on the sequence of plant community succession.     

Entomopathogenic fungi and insect behaviour: from unsuspecting hosts to targeted vectors

The behavioural response of an insect to a fungal pathogen will have a direct effect on the efficacy of the fungus as a biological control agent. In this paper we describe two processes that have a significant effect on the interactions between insects and entomopathogenic fungi: (a) the ability of target insects to detect and avoid fungal pathogens and (b) the transmission of fungal pathogens between host insects. The behavioural interactions between insects and entomopathogenic fungi are described for a variety of fungal pathogens ranging from commercially available bio-pesticides to non-formulated naturally occurring pathogens. The artificial manipulation of insect behaviour using dissemination devices to contaminate insects with entomopathogenic fungi is then described. The implications of insect behaviour on the use of fungal pathogens as biological control agents are discussed.     

Lipid composition of Cunninghamella elegans cultivated on n-alkanes]

The ability to oxidize n-alkanes was studied with various species of fungi belonging to the Cunninghamella genus. These fungi are able to assimilate hydrocarbons and to accumulate up to 1.5 g/litre of biomass. The most active strain was Cunninghamella elegans (-) 1204. The amount of lipids formed, and their composition, depended on the length of the carbon chain of oxidized alkane. The content of fat in the cells increased with the length of the hydrocarbon chain. The following lipid fractions have been detected: phospholipids, monoglycerides, diglycerides, triglycerides, sterols, free fatty acids, sterol esters, and hydrocarbons. The qualitative composition of the fractions depended, to a considerable extent, on the n-alkane utilized. Investigation of the fatty-acid composition of intracellular lipids has shown that fatty acids with an even number of carbon atoms are formed from hydrocarbons with an even number of these atoms, while fatty acids both with an even and odd number of carbon atoms are synthesized from hydrocarbons with an odd number of these atoms. The relative content of the acids with the same number of carbon atoms as that of the alkane being utilized increased with the length of the carbon chain.     

Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.     

A method to construct cDNA library of the entomopathogenic fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>, in the hemolymph of the infected locust

A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using Blastx; no selected sequences had significant similarity with insects’ genes in comparison with the data of GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR. This method may be applied to other pathogenic fungi and their host animals.     

Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease?

Plant cells engage in mutualistic and parasitic endosymbioses with a wide variety of microorganisms, ranging from Gram-negative (Rhizobium, Nostoc) and Gram-positive bacteria (Frankia), to oomycetes (Phytophthora), Chytridiomycetes, Zygomycetes (arbuscular mycorrhizal fungi) and true fungi (Erysiphe, ascomycete; Puccinia, basidiomycete). Endosymbiosis is characterised by the 'symbiosome', a compartment within host cells in which the symbiotic microorganism is either partially or completely enclosed by a host-derived membrane. The analysis of plant mutants indicates that the genetic requirements for the interaction with rhizobia and arbuscular mycorrhiza fungi are partially overlapping. The extent to which plants use similar or identical developmental programs for the intracellular accommodation of different microorganisms is, however, not clear. For example, plant cells actively weaken their cell wall to facilitate bacterial colonisation, whereas penetration by fungal symbionts appears not to be assisted in this manner. Moreover, different transport requirements are imposed on the symbiotic interface of different interactions indicating that additional system-specific components are likely to exist.     

Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest

Our understanding of how saprotrophic and mycorrhizal fungi interact to re-circulate carbon and nutrients from plant litter and soil organic matter is limited by poor understanding of their spatiotemporal dynamics. In order to investigate how different functional groups of fungi contribute to carbon and nitrogen cycling at different stages of decomposition, we studied changes in fungal community composition along vertical profiles through a Pinus sylvestris forest soil. We combined molecular identification methods with 14C dating of the organic matter, analyses of carbon:nitrogen (C:N) ratios and 15N natural abundance measurements. Saprotrophic fungi were primarily confined to relatively recently (< 4 yr) shed litter components on the surface of the forest floor, where organic carbon was mineralized while nitrogen was retained. Mycorrhizal fungi dominated in the underlying, more decomposed litter and humus, where they apparently mobilized N and made it available to their host plants. Our observations show that the degrading and nutrient-mobilizing components of the fungal community are spatially separated. This has important implications for biogeochemical studies of boreal forest ecosystems.     

SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS / ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS / ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments.

PRACTICAL APPLICATIONS

Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and R. solani in agriculturally important plant diseases.     

Fatty acid transport protein 4 is the principal very long chain fatty acyl-CoA synthetase in skin fibroblasts

Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54-64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K+ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).     

Interactions of antifungal plant defensins with fungal membrane components

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K⁺ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).