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.     

Analysis of gene expression in the vegetative and fructification phases of the white truffle, Tuber borchii Vittad., by mRNA differential display

The mRNA differential display technique was used to compare mRNA populations from fruit body and mycelium of a white truffle species in the attempt to identify and clone differentially expressed genes. The differential expression of five out of 30 amplicons was confirmed. One fragment (Tbm 56) corresponded to a part of the ribosomal genes. Three cDNA fragments (Tbf 12, Tbf 20, Tbf 21) were expressed only in the fructification phase, while the other cDNA (Tbf 55) was expressed strongly in fruit body and also detectable in the mycelium. These clones correspond to part of the single-copy genes in the Tuber borchii Vittad. genome.     

Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD

To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 alpha-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.     

Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.     

Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits

The ripening of strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, is a complex developmental process that involves many changes in gene expression. To understand how these changes relate to the biochemistry and composition of the fruit the specific genes involved have been examined. A high-quality cDNA library prepared from ripe strawberry fruit was differentially screened for ripening-related clones using cDNA from ripe and white fruits. From 112 up-regulated clones obtained in the primary screen, 66 differentially expressed clones were isolated from the secondary screen. The partial sequences of these cDNAs were compared with database sequences and 26 families of non-redundant clones were identified. Northern analysis confirmed that all of these cDNAs were ripening-enhanced. The expression of many of their corresponding genes was negatively regulated in auxin-treated fruit. These sequences, several of which are novel to fruits, encode proteins involved in key metabolic events including anthocyanin biosynthesis, cell wall degradation, sucrose and lipid metabolism, protein synthesis and degradation, and respiration. These findings are discussed in relation to the role of these genes in determining fruit quality characteristics. Received: 19 January 1998 / Accepted: 5 February 1998     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.     

Verticillium disease or "dry bubble" of cultivated mushrooms: the Agaricus bisporus lectin recognizes and binds the Verticillium fungicola cell wall glucogalactomannan

The step of recognition and (or) binding for the development of the disease of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola was studied by several approaches: agglutination of V. fungicola germinated spores by an A. bisporus extract from fruit body cell walls, immunofluorescence microscopy of A. bisporus hyphae from fruit bodies and vegetative mycelia pretreated with purified V. fungicola cell wall glucogalactomannan, and finally, by hemagglutination experiments carried out with an A. bisporus fruit body lectin in the presence and absence of the same glucogalactomannan. Hemagglutinating activity of the purified A. bisporus fruit body lectin was clearly inhibited by the V. fungicola glucogalactomannan, whereas in the A. bisporus vegetative mycelium such lectin was not encountered. All the results obtained make evident the recognition and binding of the A. bisporus fruit body lectin to the V. fungicola cell wall glucogalactomannan, clarifying why the mushrooms, but not the vegetative mycelium, become diseased.     

Isolation and characterization of some mycelia inhabiting Tuber ascomata

Tuber spp. are ectomycorrhizal ascomycetes that produce subterranean ascomata known as truffles. Truffles can be regarded as complex microhabitats hosting bacteria and yeasts. In this paper we show that guest filamentous fungi are also associated to truffle ascomata, regardless of the Tuber spp., and report the morpho-molecular characterization of seven truffle-hosted mycelia isolated from healthy and intact Tuber ascomata. Some of these isolates were shown to be related to the fungal endophytes of plants. Interestingly, the truffle-hosted mycelia grew stuck to the hyphal wall of their partner when co-cultivated with the Tuber borchii mycelium, but not when co-cultivated with the test species Agaricus macrosporus. The present data suggest that guest filamentous fungi can be added to the list of truffle-interacting microorganisms.     

Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization

Capsaicinoids responsible for pungency of chili pepper are synthesized exclusively in the placenta tissue of the fruit. As an elementary step in the molecular genetics study of capsaicinoid biosynthesis, a cDNA library was constructed from the placenta of a highly pungent pepper, Capsicum chinense cv. Habanero using the suppression subtractive hybridization (SSH). Thirty-nine cDNA clones from about 400 subtracted clones were selected through dot blot analysis and according to their nucleotides sequence. Sequence information of the chosen clones was evaluated by comparing it with DNA and protein databases. Results showed that the cDNA clones could be divided into 4 groups; cDNAs with similarities in genes encoding metabolic enzymes including acyl transferase and fatty acid alcohol oxidase (Group I), putative cell wall proteins (Group II), biotic and abiotic stress-inducible proteins (Group III), and lastly, cDNAs with no similarity (Group IV). Northern blot analysis was performed to confirm that these clones are differentially expressed in pungent pepper. The results revealed that all cDNA clones were differentially expressed in pungent pepper. In addition, the cDNA clones of Groups I and IV were differentially or preferentially expressed in the placenta of pungent pepper.     

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

Tyrosinase expression during black truffle development: from free living mycelium to ripe fruit body

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.