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.
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter
In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.
The ESTuber database () includes 3,271 Tuber borchii expressed sequence tags (EST). The dataset consists of 2,389 sequences from an in-house prepared cDNA library from truffle vegetative hyphae, and 882 sequences downloaded from GenBank and representing four libraries from white truffle mycelia and ascocarps at different developmental stages. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts. Data were collected in a MySQL database, which can be queried via a php-based web interface.
Sequences included in the ESTuber db were clustered and annotated against three databases: the GenBank nr database, the UniProtKB database and a third in-house prepared database of fungi genomic sequences. An algorithm was implemented to infer statistical classification among Gene Ontology categories from the ontology occurrences deduced from the annotation procedure against the UniProtKB database. Ontologies were also deduced from the annotation of more than 130,000 EST sequences from five filamentous fungi, for intra-species comparison purposes.
Further analyses were performed on the ESTuber db dataset, including tandem repeats search and comparison of the putative protein dataset inferred from the EST sequences to the PROSITE database for protein patterns identification. All the analyses were performed both on the complete sequence dataset and on the contig consensus sequences generated by the EST assembly procedure.
The resulting web site is a resource of data and links related to truffle expressed genes. The Sequence Report and Contig Report pages are the web interface core structures which, together with the Text search utility and the Blast utility, allow easy access to the data stored in the database.
Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.
Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and ∼13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator.
Fungi are a morphologically and physiologically diverse group of organisms with huge impacts on nearly all ecosystems. In recent years, genomes of many fungal species have been sequenced and have greatly improved our understanding of fungal biology. Ascomycetes are the largest fungal group with the highest number of sequenced genomes; however, for the Pezizales, an early-diverging lineage of filamentous ascomycetes, only one genome has been sequence to date, namely that of the black truffle. While truffles are among the most valuable edible fungi, they have a specialized life style as plant symbionts producing belowground fruiting bodies; thus it is difficult to draw conclusions about basal ascomycetes from one truffle genome alone. Therefore, we have sequenced the genome and several transcriptomes of the basal ascomycete Pyronema confluens, which has a saprobic life style typical of many ascomycetes. Comparisons with other fungal genomes showed that P. confluens has two conserved mating type genes, but that the genomic environment of the mating type genes is different from that of higher ascomycetes. We also found that a high number of orphan genes, i.e. genes without homologs in other fungi, are upregulated during sexual development. This is consistent with rapid evolution of sex-associated genes.
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.
Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form.
We report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response.
T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production.
• Background and Aims Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized.
• Methods The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT–PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2).
• Key Results The CcDH1 and CcDH2 genes encode Y3SK2 dehydrins and the CcDH3 gene encodes an SK3 dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized.
• Conclusions cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.
Dehydrins; late embryogenic abundant protein (LEA); seed development; Coffea; C. canephora; C. arabica; Rubiaceae
Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.
abiotic stress; dehydration stress; drought; cold acclimation; freezing tolerance; LEA proteins; dehydrins
Aroma variability in truffles has been attributed to maturation (Tuber borchii), linked to environmental factors (Tuber magnatum), but the involvement of genetic factors has been ignored. We investigated aroma variability in Tuber uncinatum, a species with wide distribution. Our aim was to assess aroma variability at different spatial scales (i.e. trees, countries) and to quantify how aroma was affected by genotype, fruiting body maturity, and geographical origin.A volatile fingerprinting method was used to analyze the aroma of 223 T. uncinatum fruiting bodies from seven European countries. Maturity was estimated from spore melanization. Genotypic fingerprinting was performed by amplified fragment length polymorphism (AFLP).Discriminant analysis revealed that, regardless of the geographical origin of the truffles, most of the aroma variability was caused by eight-carbon-containing volatiles (C8-VOCs). In an orchard of T. uncinatum, truffles producing different concentrations of C8-VOCs clustered around distinct host trees. This clustering was not associated with maturity, but was associated with fungal genotype.These results indicate that the variation in C8-VOCs in truffles is most likely under genetic control. They exemplify that understanding the factors behind aroma variability requires a holistic approach. Furthermore, they also raise new questions regarding the ecological role of 1-octen-3-ol in truffles.
1-octen-3-ol; amplified fragment length polymorphism (AFLP); aroma variability; truffle genetics; Tuber borchii; Tuber spp.; Tuber uncinatum; volatile organic compounds (VOCs)
Transcriptional activation of the VERNALIZATION1 gene mediates the acceleration of flowering by prolonged cold (vernalization) in temperate cereals. This study examined the earliest stages of the transcriptional response of VRN1 to low temperatures. Time-course analyses, using a sensitive quantitative PCR assay, showed that in sprouting barley seedlings VRN1 transcripts begin to accumulate within 24 hours of the onset of cold. The kinetics of the initial transcriptional response of VRN1 to cold was similar to the cold-induced genes DEHYDRIN5 (DHN5) and COLD REGULATED 14B (COR14B), but occurred at lower levels compared to cold acclimation genes or the response to longer cold treatments. Temperatures between 15 and –2 °C induced expression of VRN1 within 24 hours, with a maximal response observed between 2 and –2 °C. Transcriptional induction was also observed in undifferentiated callus cells. There were significant increases in histone acetylation levels at the VRN1 locus in response to 24-hour cold treatment. Sodium butyrate, a histone deacetylation inhibitor, triggered an increase in histone acetylation at VRN1 chromatin and elevated VRN1 transcript levels. The transcriptional response of VRN1 to short-term cold treatment was examined in near-isogenic lines that have different VRN1 genotypes, showing that an allele of the barley VRN1 gene with an insertion in the first intron and high basal expression levels has a reduced transcriptional response to short term cold treatment. This study suggests that low-temperature induction of VRN1 is a cellular response to cold triggered by the same mechanisms that mediate low-temperature induction of cold acclimation genes.
Chromatin; cereal; cold acclimation; MADS box; vernalization; VRN1.
Members of the cyanovirin-N homolog (CVNH) lectin family are found in bacteria, fungi and plants. As part of our ongoing work on CVNH structure-function studies, we determined the high-resolution NMR solution structure of the homolog from the wheat head blight disease causing ascomycetous fungus Gibberella zeae (or Fusarium graminearum), hereafter called GzCVNH. Like cyanovirin-N (CV-N), GzCVNH comprises two tandem sequence repeats and the protein sequence exhibits 30% identity with CV-N. The overall structure is similar to those of other members of the CVNH family, with the conserved pseudo-symmetric halves of the structure, domains A and B, closely resembling recently determined structures of Tuber borchii, Neurospora crassa and Ceratopteris richardii CVNH proteins. Although GzCVNH exhibits a similar glycan recognition profile to CV-N and specifically binds to Manα(1–2)Manα, its weak carbohydrate binding affinity to only one binding site is insufficient for conferring anti-HIV activity.
NMR solution structure; CVNH; lectin; antiviral protein; HIV
The development of Tuber melanosporum mycorrhizal symbiosis is associated with the production of an area devoid of vegetation (commonly referred to by the French word ‘brûlé’) around the symbiotic plants and where the fruiting bodies of T. melanosporum are usually collected. The extent of the ecological impact of such an area is still being discovered. While the relationship between T. melanosporum and the other fungi present in the brûlé has been assessed, no data are available on the relationship between this fungus and the bacteria inhabiting the brûlé.
We used DGGE and DNA microarrays of 16S rRNA gene fragments to compare the bacterial and archaeal communities inside and outside of truffle brûlés. Soil samples were collected in 2008 from four productive T. melanosporum/Quercus pubescens truffle-grounds located in Cahors, France, showing characteristic truffle brûlé. All the samples were analyzed by DGGE and one truffle-ground was analyzed also using phylogenetic microarrays. DGGE profiles showed differences in the bacterial community composition, and the microarrays revealed a few differences in relative richness between the brûlé interior and exterior zones, as well as differences in the relative abundance of several taxa.
The different signal intensities we have measured for members of bacteria and archaea inside versus outside the brûlé are the first demonstration, to our knowledge, that not only fungal communities, but also other microorganisms are affected by T. melanosporum. Firmicutes (e.g., Bacillus), several genera of Actinobacteria, and a few Cyanobacteria had greater representation inside the brûlé compared with outside, whereas Pseudomonas and several genera within the class Flavobacteriaceae had higher relative abundances outside the brûlé. The findings from this study may contribute to future searches for microbial bio-indicators of brûlés.
Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.
All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls - from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes.
All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response.
Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.
This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.
Estuarine salinity gradients are known to influence plant, bacterial and archaeal community structure. We sequenced 18S rRNA genes to investigate patterns in sediment fungal diversity (richness and evenness of taxa) and composition (taxonomic and phylogenetic) along an estuarine salinity gradient. We sampled three marshes—a salt, brackish and freshwater marsh—in Rhode Island. To compare the relative effect of the salinity gradient with that of plants, we sampled fungi in plots with Spartina patens and in plots from which plants were removed 2 years prior to sampling. The fungal sediment community was unique compared with previously sampled fungal communities; we detected more Ascomycota (78%), fewer Basidiomycota (6%) and more fungi from basal lineages (16%) (Chytridiomycota, Glomeromycota and four additional groups) than typically found in soil. Across marshes, fungal composition changed substantially, whereas fungal diversity differed only at the finest level of genetic resolution, and was highest in the intermediate, brackish marsh. In contrast, the presence of plants had a highly significant effect on fungal diversity at all levels of genetic resolution, but less of an effect on fungal composition. These results suggest that salinity (or other covarying parameters) selects for a distinctive fungal composition, and plants provide additional niches upon which taxa within these communities can specialize and coexist. Given the number of sequences from basal fungal lineages, the study also suggests that further sampling of estuarine sediments may help in understanding early fungal evolution.
community composition; estuary; fungi; ribosomal RNA; salinity
Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp.
1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression.
Dehydrins; Embryo development; Mangroves; Rhizophora mucronata; Semi-vivipary
Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.
The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.
plant genome; cis-acting promoter elements; cold; dehydration; microarray
Dothideaceous black yeast-like fungi (BYF) are known to synthesise
DHN-melanin that is inhibited by the systemic fungicide tricyclazole. The
final step of the DHN melanin pathway is the conjoining of 1,8-DHN molecules
to form the melanin polymer. There are several candidate enzymes for this
step, including phenoloxidases such as tyrosinase and laccases, peroxidases,
and perhaps also catalases. We analysed the type polyphenoloxidases that are
involved in biosynthesis of BYF melanins. For that purpose we used substrates
of o-diphenoloxidases (EC 126.96.36.199.): 4-hydroxyphenyl-pyruvic acid,
L-β-phenyllactic acid, tyrosine, pyrocatechol, 3,4-dihydroxyphenylalanine
and homogentisic acid, as well as substrates of p-diphenoloxidases (EC
188.8.131.52.): syringaldazine, resorcinol, p-phenylenediamine, phloroglucinol,
guaiacol and pyrogallic acid. Fourteen strains of black yeasts originating
from different natural biotopes were investigated. The tested strains could be
divided into four groups based on their ability to produce dark pigments when
cultivated on aromatic substrates of o- and on p-diphenoloxidases. It was
established that syringaldazine, pyrogallic acid and 4-hydrophenyl-pyruvic
acid, β-phenyllactic acid optimally promote melanin biosynthesis. Average
intensity of pigmentation of all strains studied was minimal when guaiacol was
used as a substrate. The present investigation indicates that the melanisation
process may involve more enzymes and more substrates than those commonly
recognised. Black yeasts are likely to contain a multipotent
Black yeast-like fungi; Dothideales; dothideaceous black yeasts; 1,8-dihydroxynaphthalene-melanin; phenoloxidases; o-diphenoloxidases; p-diphenoloxidases
Tuber melanosporum is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production yields are often lower than expected. We assessed the genetic diversity of T. melanosporum with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus. Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey of nursery seedlings and trees inoculated with T. melanosporum revealed that <70% of seedlings and host trees were colonized with T. melanosporum and that some trees had been contaminated by Tuber brumale, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence of both mating types on host trees.