Sulfate metabolism in Tuber borchii: characterization of a putative sulfate transporter and the homocysteine synthase genes.

The homocysteine synthase (tbhos) and putative sulfate transporter (tbsul1) genes have been characterized in order to understand the sulfate metabolism and regulation in the ectomycorrhizal fungus Tuber borchii. The analyses of tbsul1 and tbhos nucleotide and deduced amino acid sequences led to the identification of the typical domains shown in homologous proteins. Sulfate starvation condition upregulates both genes. The real-time PCR assay of tbsul1 revealed that gene expression was about threefold higher in mycelia grown under sulfate starvation for 2 days than in the mycelial control and in the same starvation condition, the sulfate uptake increased. Real-time PCR and enzymatic assays showed regulation of tbhos when sulfur sources were lacking, suggesting that a transcriptional regulation of this gene rather than a post-transcriptional one occurred. Furthermore, the tbsul1 and tbhos expression patterns were evaluated during the truffle life cycle, revealing an over-expression in the mature ascomata for both genes. In the ectomycorrhizal tissue, only tbhos was upregulated suggesting its substantial role in T. borchii cysteine synthesis. The regulation of tbsul1 and tbhos occurs primarily at the transcriptional level both during vegetative and fruiting phases and these genes could be directly involved in VOCs production.

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida.

Abstract Cobalamin (vitamin B(12)) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B(12). A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B(12)-binding protein precursor of P. profundum, 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida, but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida, the recombinant protein was expressed with a C-terminal His(6)-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 +/- 2 microm.

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.

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.

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.

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.

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 interinstitutional development of an international health course.

An interinstitutional project to develop a self-instructional course in international health is described. Included is the rationale which supported the creation of the project and the operating procedures used to produce the course materials. The design and results of a field trial which provided the evaluative feedback for revising course materials are also described. The conclusion offers specific practical recommendations for the interinstitutional development of instructional materials and for the utilization of the course materials produced in this project.