Truffle Tuber Borchii Research Articles

From Nontargeted to Targeted Analysis: Feature Selection in the Differentiation of Truffle Species (Tuber spp.) Using 1H NMR Spectroscopy and Support Vector Machine.

The price of different truffle types varies according to their culinary value, sometimes by more than a factor of 10. Nonprofessionals can hardly distinguish visually the species within the white or black truffles, making the possibility of food fraud very easy. Therefore, the identification of different truffle species (Tuber spp.) is an analytical task that could be solved in this study. The polar extract from a total of 80 truffle samples was analyzed by 1H NMR spectroscopy in combination with chemometric methods covering five commercially relevant species. All classification models were validated applying a repeated nested cross-validation. In direct comparison, the two very similar looking and closely related black representatives Tuber melanosporum and Tuber indicum could be classified 100% correctly. The most expensive truffle Tuber magnatum could be distinguished 100% from the other relevant white truffle Tuber borchii. In addition, signals for a potential Tuber borchii and a potential Tuber melanosporum marker for targeted approaches could be detected, and the corresponding molecules were identified as betaine and ribonate. A model covering all five truffle species Tuber aestivum, Tuber borchii, Tuber indicum, Tuber magnatum, and Tuber melanosporum was able to correctly discriminate between each of the species.

Improving truffle mycelium flavour through strain selection targeting volatiles of the Ehrlich pathway

Truffles (Tuber spp.) are the fruiting bodies of symbiotic fungi, which are prized food delicacies. The marked aroma variability observed among truffles of the same species has been attributed to a series of factors that are still debated. This is because factors (i.e. genetics, maturation, geographical location and the microbial community colonizing truffles) often co-vary in truffle orchards. Here, we removed the co-variance effect by investigating truffle flavour in axenic cultures of nine strains of the white truffle Tuber borchii. This allowed us to investigate the influence of genetics on truffle aroma. Specifically, we quantified aroma variability and explored whether strain selection could be used to improve human-sensed truffle flavour. Our results illustrate that aroma variability among strains is predominantly linked to amino acid catabolism through the Ehrlich pathway, as confirmed by 13C labelling experiments. We furthermore exemplified through sensory analysis that the human nose is able to distinguish among strains and that sulfur volatiles derived from the catabolism of methionine have the strongest influence on aroma characteristics. Overall, our results demonstrate that genetics influences truffle aroma much more deeply than previously thought and illustrate the usefulness of strain selection for improving truffle flavour.

Bacteria associated with truffle-fruiting bodies contribute to truffle aroma.

Truffles, symbiotic fungi renown for the captivating aroma of their fruiting bodies, are colonized by a complex bacterial community of unknown function. We characterized the bacterial community of the white truffle Tuber borchii and tested the involvement of its microbiome in the production of sulphur-containing volatiles. We found that sulphur-containing volatiles such as thiophene derivatives, characteristic of T. borchii fruiting bodies, resulted from the biotransformation of non-volatile precursor(s) into volatile compounds by bacteria. The bacterial community of T. borchii was dominated by α- and β-Proteobacteria. Interestingly, all bacteria phyla/classes tested in this study were able to produce thiophene volatiles from T. borchii fruiting body extract, irrespective of their isolation source (truffle or other sources). This indicates that the ability to produce thiophene volatiles might be widespread among bacteria and possibly linked to primary metabolism. Treatment of fruiting bodies with antibacterial agents fully suppressed the production of thiophene volatiles while fungicides had no inhibitory effect. This suggests that during the sexual stage of truffles, thiophene volatiles are exclusively synthesized by bacteria and not by the truffle. At this stage, the origin of thiophenes precursor in T. borchii remains elusive and the involvement of yeasts or other bacteria cannot be excluded.

Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

Truffles regulate plant root morphogenesis via the production of auxin and ethylene.

Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction, i.e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle Tuber melanopsorum induce alterations in root morphology of the host Cistus incanus and the nonhost Arabidopsis (Arabidopsis thaliana; i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and IAA fully mimicked the root morphology induced by the mycelium for both host and nonhost plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene-insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungus-induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffles synthesize ethylene from l-methionine probably through the alpha-keto-gamma-(methylthio)butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions.

A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins

BackgroundThe expressed sequence tag M6G10 was originally isolated from a screening for differentially expressed transcripts during the reproductive stage of the white truffle Tuber borchii. mRNA levels for M6G10 increased dramatically during fruiting body maturation compared to the vegetative mycelial stage.ResultsBioinformatics tools, phylogenetic analysis and expression studies were used to support the hypothesis that this sequence, named TbDHN1, is the first dehydrin (DHN)-like coding gene isolated in fungi. Homologs of this gene, all defined as "coding for hypothetical proteins" in public databases, were exclusively found in ascomycetous fungi and in plants. Although complete (or almost complete) fungal genomes and EST collections of some Basidiomycota and Glomeromycota are already available, DHN-like proteins appear to be represented only in Ascomycota. A new and previously uncharacterized conserved signature pattern was identified and proposed to Uniprot database as the main distinguishing feature of this new group of DHNs. Expression studies provide experimental evidence of a transcript induction of TbDHN1 during cellular dehydration.ConclusionExpression pattern and sequence similarities to known plant DHNs indicate that TbDHN1 is the first characterized DHN-like protein in fungi. The high similarity of TbDHN1 with homolog coding sequences implies the existence of a novel fungal/plant group of LEA Class II proteins characterized by a previously undescribed signature pattern.

Transcript Profiling Reveals Novel Marker Genes Involved in Fruiting Body Formation in Tuber borchii

cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle Tuber borchii. Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.

The anti‐HIV cyanovirin‐N domain is evolutionarily conserved and occurs as a protein module in eukaryotes

A novel protein family homologous to the sugar-binding antiviral protein cyanovirin-N (CVN) is described. CVN, an 11-kDa protein that, by binding to the high-mannose moiety of certain viral surface glycoproteins, blocks virus entry into target cells, has thus far been identified only in the cyanobacterium Nostoc ellipsosporum. Here we show that CVN belongs to a protein family identified by analysis of transcript sequences deriving from a gene expression profiling study conducted in the truffle Tuber borchii. Members of this family (named CyanoVirin-N Homology) are found in filamentous ascomycetes and in the fern Ceratopteris richardii. As revealed by 3D structure-based searches, all CVNH proteins have a predicted fold that matches the so far unique fold of the cyanobacterial polypeptide. The CVNH domain is a versatile protein module. In ferns and cyanobacteria it is found in secretory proteins. In filamentous ascomycetes it is found in nonsecretory monodomain proteins as well as part of multidomain proteins bearing functionally related modules such as the peptidoglycan and chitin-binding domain LysM. Transcript abundance data further indicate that the expression of different CVNH forms is modulated in response to nutrient availability. These findings have implications for the understanding of protein-oligosaccharide interaction in fungi and plants, and provide candidate polypeptides to be tested and exploited as antiviral agents.

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii.

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus OUT/C terminus IN orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K m for ammonium of 2 μM . The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.

Cloning, Expression, and Characterization of the hxk-1 Gene from the White Truffle Tuber borchii Vittad.: A First Step toward Understanding Sugar Metabolism

Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HKM1, HKM2, and HKM3). In the investigation described here, a gene coding for hexokinase (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized. The Kcat value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.

Temperature-dependent evolution of volatile organic compounds inTuber borchii from Italy

The concentrations of the volatile organic compounds of the truffle Tuber borchii have been determined by gas solid extraction and purge and trap injection in GC–MS, and their variations were observed during storage. In fresh samples 1-octen-3-ol is the main volatile product, together with lower amounts of alcohols, aldehydes and 2- and 3-methylthiophenes as the only sulphur compounds. The release of (E)-2-pentenal is the most important degradation pattern at low temperatures, while bis-3-methylbutyl disulphide, 2-methyl-4,5-dihydrothiophene and 3-methyl-4,5-dihydrothiophene are evolved at room temperature. Copyright © 2001 John Wiley & Sons, Ltd.