Aminotenuazonic Acid: Isolation, Structure Elucidation, Total Synthesis and Herbicidal Activity of a New Tetramic Acid from Fruiting Bodies of Laccaria Species.

(5S,6S)-Aminotenuazonic acid, a new 3-acyltetramic acid, related to the well-known mycotoxin tenuazonic acid has been isolated from fruiting bodies of Laccaria bicolor. Its structure was mostly established by analysis of its 2D NMR and HR-(+)-ESI-MS spectra. A total synthesis starting from N-Boc-l-isoleucine gave (5S,6S)-aminotenuazonic acid in 8 % yield over nine steps (67 % de). The key steps of the total synthesis are a light-initiated Hofmann-Löffler-Freytag radical chain reaction and a Dieckmann cyclisation. The relative and absolute configurations of the natural product were determined by comparison of its NMR and CD spectra with those of the corresponding enantiopure synthetic compounds. Metabolic profiling of crude extracts of different mushrooms showed that aminotenuazonic acid is present in all four of the investigated Laccaria species. Aminotenuazonic acid shows phytotoxic activities against the root and shoot growth of Lepidium sativum, Pinus sylvestris and Arabidopsis thaliana comparable to those of tenuazonic acid.

Physicochemical and in vitro cytotoxic properties of chitosan from mushroom species (Boletus bovinus and Laccaria laccata).

Chitosan samples from two mushroom species (Boletus bovinus, Laccaria laccata) were obtained and characterized by viscosimetry, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), elemental analyses (EA), nuclear magnetic resonance spectroscopy (13C NMR), X-ray diffraction (XRD) and thermogravimetric (TGA) analyses. Properties of the fungal chitosan samples were compared to commercial low-molecular weight chitosan, crustacean chitosan (Cervimunida johni) and chitosan obtained from an insect (Hilobius abietis). Additionally, the cytotoxic properties of chitosan in vitro on cancerous hepatoma and non-cancerous ovary cells cultivated on films with different chitosan concentrations was evaluated. As a conclusion, this study clearly revealed that low-molecular weight chitosan films and solutions with high degree of deacetylation can act cytotoxically on both tumor MH-22A and normal CHO cells in vitro. Consequently, this work may be useful for further investigations of natural anticancer products in medical areas.

Nematicidal anthranilic acid derivatives from Laccaria species.

Three undescribed natural products, the anthranilic acid derivatives laccanthrilic acids A, B, and C, as well as the known (3S)-1,2,3,4-tetrahydro-3-ß-carboline-3-carboxylic acid were isolated from fruiting bodies of Laccaria laccata. The structures were established by 1D and 2D NMR spectroscopy, HR-(+)-ESIMS and chemical synthesis. The absolute configuration of laccanthrilic acids A and B was determined by GC-MS after hydrolytic cleavage and derivatisation of the resulting glutamic acid with methanol and Mosher's reagent and subsequent comparison with authentic synthetic samples of known absolute configuration. The absolute configuration of laccanthrilic acid C was determined by comparison of the CD spectra of laccanthrilic acids B and C with each other. Metabolic profiling of related species showed that the compounds are common in the genus Laccaria. Laccanthrilic acid B exhibited moderate nematicidal effects against Caenorhabditis elegans, which might explain to some degree the beneficial role of these fungi for the growth and survival of their host plants.

Crystal Structures of Fungal Tectonin in Complex with O-Methylated Glycans Suggest Key Role in Innate Immune Defense.

Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction.

DeltaMS: a tool to track isotopologues in GC- and LC-MS data.

INTRODUCTION: Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved in metal complexing. OBJECTIVE: To introduce a software tool for the identification of isotopologues from mass spectrometry data. METHODS: DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) search for isotope signatures with a specific mass shift and intensity ratio in one sample set, (2) analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not, (3) analyze isotope-guided perturbation experiments with a setup described in X13CMS. RESULTS: To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures. CONCLUSION: DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.

Arsenic concentrations and associated health risks in Laccaria mushrooms from Yunnan (SW China).

Some species of Laccaria have been known to contain relatively high levels of arsenic in Europe and are used as edible mushrooms in the southwest China. One population of Laccaria proxima and one population of L. vinaceoavellanea as well as topsoil (0-10 cm) they grew on were collected from natural habitats of Yunnan (SW China), while other samples such as Laccaria mushroom samples without soil were purchased from four different local markets in Yunnan. Concentrations of arsenic were determined in fruit bodies of the mushrooms and in the soils by using atomic fluorescence spectrometry to assess potential health risks of these species. The mean arsenic concentrations in caps were 135, 14.1-143, 5.5 and 130-163 mg kg(-1) dry weight (dw) for Laccaria amethystina, Laccaria laccata, L. proxima and L. vinaceoavellanea, respectively. The mean value for bioconcentration factor of arsenic in caps of L. vinaceoavellanea was 29.1 for soil with arsenic content at 5.6 mg kg(-1) dw, which indicate that L. vinaceoavellanea is an accumulator for arsenic. Caps of L. amethystina, L. laccata and L. vinaceoavellanea consumed at a volume of 300 g fresh weight for a single meal in a week can yield an exposure amount of arsenic at 4.1, 0.42-4.3 and 3.9-4.9 mg, respectively. These values are higher than the limit dose for the intake of inorganic arsenic recommended by the Joint FAO/WHO Expert Committee on Food Additives.

A secreted effector protein of Laccaria bicolor is required for symbiosis development.

Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.

Survey and analysis of simple sequence repeats in the Laccaria bicolor genome, with development of microsatellite markers.

It is becoming clear that simple sequence repeats (SSRs) play a significant role in fungal genome organization, and they are a large source of genetic markers for population genetics and meiotic maps. We identified SSRs in the Laccaria bicolor genome by in silico survey and analyzed their distribution in the different genomic regions. We also compared the abundance and distribution of SSRs in L. bicolor with those of the following fungal genomes: Phanerochaete chrysosporium, Coprinopsis cinerea, Ustilago maydis, Cryptococcus neoformans, Aspergillus nidulans, Magnaporthe grisea, Neurospora crassa and Saccharomyces cerevisiae. Using the MISA computer program, we detected 277,062 SSRs in the L. bicolor genome representing 8% of the assembled genomic sequence. Among the analyzed basidiomycetes, L. bicolor exhibited the highest SSR density although no correlation between relative abundance and the genome sizes was observed. In most genomes the short motifs (mono- to trinucleotides) were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. In the L. bicolor genome, most of the SSRs were located in intergenic regions (73.3%) and the highest SSR density was observed in transposable elements (TEs; 6,706 SSRs/Mb). However, 81% of the protein-coding genes contained SSRs in their exons, suggesting that SSR polymorphism may alter gene phenotypes. Within a L. bicolor offspring, sequence polymorphism of 78 SSRs was mainly detected in non-TE intergenic regions. Unlike previously developed microsatellite markers, these new ones are spread throughout the genome; these markers could have immediate applications in population genetics.

A new isoprenyl phenyl ether riboside from the culture of basidiomycete Laccaria amethystea.

A new isoprenyl phenyl ether riboside, 3-(3-methylbut-2-enyloxy)-4-O-alpha-D-ribofuranose benzoic acid methyl ester (1), was isolated from the culture of basidiomycete Laccaria amethystea. The structure of 1 was elucidated on the basis of extensive spectroscopic analysis.

Assessment of alcohol percentage test for fungal surface hydrophobicity measurement.

AIM: To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces. METHODS AND RESULTS: APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R(2) = 0.95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs <90 degrees and no measurable degree of hydrophobicity using the APT method. CONCLUSIONS: The APT method was effective in measuring the degree of hydrophobicity and can be conducted on different zones of fungal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of fungal surface hydrophobicity is important for understanding of its particular role and function in fungal morphogenesis and pathogenesis. APT is a simple method that can be utilized for fungal hydrophobicity measurements when CA cannot be measured because of obscured view from aerial mycelia growth.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.