Raffaella Pierleoni

Identification of putative genes involved in the development of Tuber borchii fruit body by mRNA differential display in agarose gel

Abstract. In order to analyse gene expression during fruit body development of the ectomychorrizal fungus Tuber borchii Vittad., a modified differential display procedure was set up. The procedure used is easier and faster than the traditional one and generates reproducible cDNA banding patterns that can be resolved on a standard ethidium bromide-agarose gel. From 16 cDNA fingerprints, 25 amplicons with apparent differential expression were identified and cloned without a previous reamplification. Fifteen clones showed significant similarity to known proteins that are involved in dikaryosis and fruiting, cell division, transport across membranes, mitochondrial division, intermediary metabolism, biosynthesis of isoprenoid compounds and putative RNA/DNA binding. Northern blot analyses confirmed that seven cDNAs were indeed differentially expressed during fruit body development. The characterisation of these cDNAs represents a starting point in understanding the molecular mechanisms of cellular differentiation leading to the development of the T. borchii fruit body.

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a 13C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.

Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos-Tuber borchii ectomycorrhizae

No information is available about Tuber borchii Vittad. ammonium metabolism during its life cycle, which involves the succession of three distinct phases. In this direction, the levels of glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13-14) and glutamate dehydrogenase (GDH; EC 1.4.1.2-4) were evaluated in Tilia platyphyllos Scop.-Tuber borchii Vittad. ectomycorrhizae, free living mycelium and non-inoculated roots. In the plant roots, GS shows high specific activity and only NADH-GDH (EC 1.4.1.2) is detectable; on the other hand, in free living mycelium GS and NADPH-GDH (EC 1.4.1.4) can be detected. Ectomycorrhizal metabolism was found to be deeply influenced by the two symbiotic partners. In fact, GS and both forms of GDH are present and their specific activities are higher than those found in the plant root and in the mycelial cells.

Identification of Tuber borchii Vittad. mycelium proteins separated by two-dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging.

This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5—9 and a molecular mass of 10—200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty‐three proteins were excised and analyzed by the combination of amino acid and N‐terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS‐PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases.

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.