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-β-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.     

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.     

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 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     

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.     

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.     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

Molecular characterization of cDNAs corresponding to genes expressed during almond (Prunus amygdalus Batsch) seed development

A number of different cDNA clones corresponding to the most abundant mRNAs present in immature seeds have been isolated from an almond (Prunus amygdalus cv. Texas) immature seed cDNA library. Those corresponding to proteins involved in storage processes have been further characterized. Two of these cDNAs (PA3BF1 and PA3BE12) code for the almond globulins (prunins), the main family of storage proteins synthesized in seeds during embryogenesis, and another cDNA (PA3BA1) codes for the 15.7 kDa almond oleosin, a protein located on the surface of oil bodies in plant seeds. These cDNAs have been sequenced and their expression during almond fruit development has been studied. Their expression is seed-specific and localized in cotyledons around 100 days after flowering. Both prunin and oleosin genes are present in one or two copies in the almond genome.     

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.     

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.     

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     

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.     

Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

Identification and expression analysis of two fungal cDNAs regulated by ectomycorrhiza and fruit body formation

Ectomycorrhiza formation is a complex developmental process that is still not well understood. To study this process, we identified genetic markers for mycorrhiza development by differential screening of a cDNA library obtained from fully developed Picea abies – Amanita muscaria mycorrhizas. Twenty-three cDNA clones were identified that showed significantly altered gene expression during the ectomycorrhizal interaction. A detailed analysis was performed for two fungal cDNA clones, SC13 and SC25, exhibiting the most pronounced differences. SC13 encodes a protein of 184 amino acid residues that shows no homology with any sequence in databases. It was highly expressed in non-mycorrhizal hyphae, whereas its expression was decreased at least 50-fold in mycorrhizas and fruit bodies. SC25 encodes a protein of 198 residues that shows weak sequence homology with extensin-like plant proteins. The expression of this gene was weak in non-mycorrhizal hyphae but approx. 30-fold higher in mycorrhizas and fruit bodies. Because the expression of both developmentally regulated fungal genes was identical for mycorrhizas and fruit bodies, a common regulation mechanism for both developmental processes is proposed.