Biomedical subjects

V Stocchi

Publications and source records attributed to V Stocchi.

At least 19 recordsLinked to original sources

PubMed2006

The expression profile of the Tuber borchii nitrite reductase suggests its positive contribution to host plant nitrogen nutrition.

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant's ability to make use of nitrate/nitrite in its nitrogen nutrition.

Amino Acid Sequence↗

PubMed2006

The isoprenoid pathway in the ectomycorrhizal fungus Tuber borchii Vittad.: cloning and characterisation of the tbhmgr, tbfpps and tbsqs genes.

The isoprenoid pathway of the ectomycorrhizal fungus Tuber borchii Vittad is investigated to better understand the molecular mechanisms at work, in particular during the maturation of the complex ascomata (the so-called "truffles"). Three T. borchii genes coding for the most important regulatory enzymes of the isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl-diphosphate synthase (FPPS) and squalene synthase (SQS), were cloned and characterised. The analyses of their nucleotide and deduced amino acid sequences led us to identify the typical domains shown in homologous proteins. By using a quantitative real-time PCR the expression pattern of the three genes was analysed in the vegetative phase and during the complex ascoma maturation process, revealing an over-expression in the mature ascomata. The enzymatic activity of the T. borchii 3-hydroxy-3-methylglutaril-CoA reductase (HMGR) was investigated with a HPLC method, confirming that the significant isoprenoid biosynthesis in ripe ascomata proceeds not only via a transcriptional activation, but also via an enzyme activity control. These findings imply that isoprenoids play a fundamental role in Tuber ascomata, particularly in the last phases of their maturation, when they could be involved in antifungal or/and antimicrobial processes and contribute to the famous flavour of the truffle ascomata.

Amino Acid Sequence↗

PubMed2006

Molecular characterisation of a Tuber borchii Smt3 gene.

Tbsmt3 gene from the ectomychorrizal fungus Tuber borchii was identified and sequenced. The Tbsmt3 gene encodes for a protein sharing significant amino acid homology with the yeast SMT3, a ubiquitin-like protein that is post-translationally attached to several proteins involved in many cellular processes. The comparison between the Tbsmt3 genomic and cDNA sequences established that the encoding sequence is interrupted by an intron of 312 bp. Southern blot analysis revealed only one copy of Tbsmt3 gene in the T. borchii genome. Tbsmt3 is expressed in all phases of T. borchii life cycle: mycelium, ectomycorrhiza and ascoma. However, the Tbsmt3 mRNA decreased during fruit body maturation.

Amino Acid Sequence↗

PubMed2006

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK.

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.

Fungal Proteins↗

PubMed2006

Characterization and biological activity of the main flavonoids from Swiss Chard (Beta vulgaris subspecies cycla).

The molecular components of a phenolic fraction (P2), obtained from liquid chromatography of a Swiss Chard (Beta vulgaris subsp. cycla) extract, were identified using HPLC-ESI-MS/MS. The primary P2 components were: vitexin-2''O-rhamnoside, its demethylated form 2''-xylosylvitexin, isorhamnetin 3-gentiobioside, and rutin. P2 "in toto" and the single components were characterized for antioxidant capacity, antimitotic activity on MCF-7 human breast cancer cells and for toxicity to human lymphocytes and macrophages. P2 inhibited MCF-7 cell proliferation (IC(50) value = 9 microg/ml) without inducing apoptosis, showed no toxicity to human lymphocytes and slight toxicity to macrophages. Vitexin-2''O-rhamnoside strongly inhibited DNA synthesis in MCF-7 cells, whereas 2''-xylosylvitexin and isorhamnetin 3-gentiobioside were activators; combinations of activators and inhibitors maintained the over-all inhibitory effect.

Antineoplastic Agents, Phytogenic↗

PubMed2005

Determination of microbial volatile organic compounds from Staphylococcus pasteuri against Tuber borchii using solid-phase microextraction and gas chromatography/ion trap mass spectrometry.

The mycelium of Tuber borchii Vittad., a commercial truffle species, is used as a model system for in vitro ectomycorrhizal synthesis, infected seedling production and biotechnological applications. Our fungal cultures were accidentally contaminated with a Staphylococcus pasteuri strain, showing a strong antifungal activity against T. borchii mycelium. In order to identify the antifungal volatile agents produced by S. pasteuri, solid-phase microextraction (SPME) with gas chromatography and mass spectrometry (GC/MS) was used. Using this method 65 microbial volatile organic compounds (MVOCs), synthesized by this bacterium in either single or in fungal-bacterial dual culture, were identified. SPME combined with GC/MS may be a useful method for the determination of MVOCs involved in the antifungal activity. These results showed that bacteria with unusual biological activities could be a major problem during large-scale production of inoculum for truffle-infected seedling.

Fungi↗

PubMed2004

Differential gene expression during pre-symbiotic interaction between Tuber borchii Vittad. and Tilia americana L.

Ectomycorrhizal formation is a highly regulated process involving the molecular reorganization of both partners during symbiosis. An analogous molecular process also occurs during the pre-symbiotic phase, when the partners exchange molecular signals in order to position and prepare both organisms for the establishment of symbiosis. To gain insight into genetic reorganization in Tuber borchii during its interaction with its symbiotic partner Tilia americana, we set up a culture system in which the mycelium interacts with the plant, even though there is no actual physical contact between the two organisms. The selected strategies, suppressive subtractive hybridisation and reverse Northern blots, allowed us to identify, for the first time, 58 cDNA clones differentially expressed in the pre-symbiotic phase. Sequence analysis of the expressed sequence tags showed that the expressed genes are involved in several biochemical pathways: secretion and apical growth, cellular detoxification, general metabolism and both mutualistic and symbiotic features.

Ascomycota↗

PubMed2003

A putative mitochondrial fission gene from the ectomycorrhizal ascomycete Tuber borchii Vittad.: cloning, characterisation and phylogeny.

Mitochondrial binary division is a complex process occurring in multiple steps, mediated by several proteins. In Saccharomyces cerevisiae, a mitochondrial membrane protein, Fis1p, is required for the proper assembly of the mitochondrial division apparatus. In this study, we report the cloning, characterisation and phylogenetic analysis of Tbfis1, a gene from the ectomycorrhizal ascomycetous truffle Tuber borchii, encoding for an orthologue of S. cerevisiae Fis1p. The Tbfis1 coding region consists of a 468-nucleotide open reading frame interrupted by four introns, which encodes for a polypeptide of 155 amino acids, having a predicted transmembrane domain structure typical of the Fis1p Family. Southern blot analysis revealed that Tbfis1 is a single-copy gene in the T. borchii genome. Tbfis1 is highly expressed during the first stages of T. borchii fruit body ripening, while its expression decreases during T. borchii mycelium ageing. Also, Virtual Northern blot analysis revealed Tbfis1 expression in the symbiotic phase of the fungus life cycle. Phylogenetic analysis allowed the identification of Tbfis1 orthologues in filamentous fungi, yeasts, plants, worms, flies and mammals, indicating that the function of the protein coded by this gene has been conserved during evolution.

Amino Acid Sequence↗

PubMed2003

Characterization of the Tuber borchii nitrate reductase gene and its role in ectomycorrhizae.

The nitrate assimilation pathway represents a useful model system in which to study the contribution of a mycorrhizal fungus to the nitrogen nutrition of its host plant. In the present work we cloned and characterized the nitrate reductase gene (tbnr1) from Tuber borchii. The coding region of tbnr1 is 2,787 nt in length, and it encodes a protein of 929 amino acids. Biochemical and Northern-blot analyses revealed that nitrate assimilation in T. borchii is an inducible system that responds mainly to nitrate. Furthermore, we cloned a nitrate reductase cDNA (tpnr1) from Tilia platyphyllos to set up a quantitative real-time PCR assay that would allow us to determine the fungal contribution to nitrate assimilation in ectomycorrhizal tissue. Using this approach we demonstrated that the level of tbnr1 expression in ectomycorhizae is eight times higher than in free-living mycelia, whereas tpnr1 transcription was found to be down-regulated after the establishment of the symbiosis. Enzymatic assays showed that NADPH-dependent nitrite formation markedly increases in ectomycorrhizae. These findings imply that the fungal partner plays a fundamental role in nitrate assimilation by ectomycorrhizae. Amino acid determination by HPLC revealed higher levels of glutamate, glutamine and asparagine in symbiotic tissues compared with mycelial controls, thus suggesting that these amino acids may represent the compounds that serve to transfer nitrogen to the host plant.

Amino Acid Sequence↗

PubMed2002

Comparative study and molecular characterization of ectomycorrhizas in Tilia americana and Quercus pubescens with Tuber brumale.

Mycorrhizas of Tuber brumale on Quercus pubescens and Tilia americana were obtained in vitro using micropropagated plantlets. Mycelium pure cultures were used for inoculation. Both the mycelium used for the inoculations, as well as the mycorrhizas which were obtained, were identified using several molecular approaches: analysis of the ITS region, polymerase chain reaction (PCR) specific primers and sequencing. The mycorrhizas were described from a morphological standpoint. Some of their biometric characteristics were different in bass-wood than they were in oak, thus showing the influence of the host plant on several of the morphological features believed to be necessary for the identification of the species. Considering the variability of their morphological characteristics, molecular analysis proved to be a necessary tool for the recognition of the mycorrhizas of Tuber spp.

Ascomycota↗

PubMed2002

Solid-phase microextraction gas chromatography/ion trap mass spectrometry and multistage mass spectrometry experiments in the characterization of germacrene D.

Germacrene D is a vegetable pheromone utilized in interactions among organisms belonging to different species. For the first time, using solid-phase microextraction/gas chromatography/ion trap mass spectrometry, the presence of this compound was detected in an in vitro mycorrhizal synthesis system where the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. interacts with the plant Tilia Americana L. From this symbiosis, a new structure, called ectomycorrhiza, is formed where the two symbionts exchange nutrients and metabolites. It seems that only after this interaction can the mycelium develop the fruitbody, commonly known as truffle. The results obtained allowed us to ascertain that germacrene D was synthesized by the plant exclusively in the presence of T. borchii. The originality of these data prompted us to hypothesize that this compound could be involved in the first step of ectomycorrhiza formation, as it is able to stimulate specific fungi receptors. In fact, plants release hundreds of secondary metabolites that are important in their interactions with other organisms.

Gas Chromatography-Mass Spectrometry↗

PubMed2001

Identification of differentially expressed cDNA clones in Tilia platyphyllos-Tuber borchii ectomycorrhizae using a differential screening approach.

No information is presently available on the molecular mechanisms that control the morphogenesis of the truffle, an ectomycorrhizal ascomycetous fungus of great economic interest not only for forestry and agronomy but also for the organoleptic properties of its hypogeous fruitbodies. A Tilia platyphyllos- Tuber borchii model system was used in order to identify genes induced or up-regulated during symbiosis, since their isolation is a prerequisite for the understanding of the molecular bases of mycorrhizal development and regulation. The strategy applied involved the construction of an ectomycorrhizal cDNA library and random selection of clones, followed by a differential screening procedure to analyse cDNA expression in uninfected roots, ectomycorrhizae and free-living mycelia. The results revealed that many genes - and more plant genes than fungal genes - are expressed at higher levels during the symbiotic phase. Several clones were also investigated in order to understand their biological function. This study represents the first attempt to extend our knowledge of the molecular mechanisms underlying the establishment of ectomycorrhiza in Tuber species.

Ascomycota↗

PubMed2001

Morphological and molecular characterisation of Pulvinula constellatio ectomycorrhizae.

The ascomycete Pulvinula constellatio was found to form competing ectomycorrhizal relationships with plants that had been inoculated with Tuber spp. and other edible ectomycorrhizal fungi. In order to be able to distinguish P. constellatio mycorrhizae from those of the Tuber spp., we determined the morphological and bio-molecular characteristics of P. constellatio. The complete sequence of the ITS regions was determined, in order to select specific primers. The ITS region was also studied using restriction fragment length polymorphism analyses with several restriction enzymes that allowed an unambiguous identification of the species.

Ascomycota↗

PubMed2001

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 (HK(M1), HK(M2), 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 K(cat) 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.

Amino Acid Sequence↗

PubMed2001

One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli.

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.

Chromatography, Affinity↗

PubMed2001

Cloning and characterisation of a polyubiquitin gene from the ectomycorrhizal fungus Tuber borchii vittad.

Ubiquitin is a highly conserved 76-amino acid protein implicated in the function of quite different vital cellular processes. In the present study, we cloned and sequenced a polyubiquitin gene from Tuber borchii (Ubil) that is organised in four tandem repeats, with two C-terminal extension amino acids, serine and leucine. Two introns of 116 bp and 55 bp in length were detected in the first and second repeats, respectively. The Ubil gene is highly expressed in mycelium and is less expressed in the ripe fruiting body. Southern and Northern blot analyses revealed a second form of the ubiquitin gene.

Amino Acid Sequence↗

PubMed2001

Effects of different carbohydrate sources on the growth of Tuber borchii Vittad. mycelium strains in pure culture.

The influence of carbohydrate utilisation on the growth of three strains of Tuber borchii Vittad. mycelium (1BO, 17BO and 10RA) in culture was assessed using culture media containing glucose (control), mannose or mannitol. Mannose was the best substrate for growth of the strains and this was particularly evident for strain 17BO. Mannitol instead was metabolized only by 10RA and 1BO. In order to explain the different growth trends, analyses of enzyme levels, kinetic parameters, protein patterns and the morphology of the three strains were carried out. Our results show that these strains of T. borchii mycelium were affected by the substrates used in the media. The aim of the present work was to optimise the in vitro production of T. borchii mycelium for use in experiments which require the fungus in precise and reproducible conditions, such as mycorrhizal synthesis or protein and nucleic acid extractions.

Ascomycota↗

PubMed2001

Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies.

Previous studies on Tuber borchii fruit bodies in early maturation stages suggested a role of bacteria in sporocarp structural modifications. In order to verify this hypothesis, in the present study we investigated by means of microbial and ultrastructural approaches, the bacterial population of T. borchii sporocarps from intermediate maturation phases to advanced decomposition stages, paying particular attention to chitinolytic and cellulolytic bacteria and to their relationships with ascii and ascospores. We found that Pseudomonas fluorescens and spore-forming Bacillaceae, both able to degrade cellulose and chitin, are present inside the sporocarps in all maturation stages investigated. Moreover, rod-shaped bacteria seem able to erode ascus walls and colonize the interior of ascii containing mature spores. These results suggest a possible role of these bacteria in the process of ascus opening. Moreover, the presence of P. fluorescens and Bacillaceae on isolated mature spores after decontamination suggests an intimate association between these bacteria and the ascospores.

Ascomycota↗