Barbara Montanini

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today’s truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a ‘symbiosis toolbox’. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ∼125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis—‘the symbiosis toolbox’—evolved along different ways in ascomycetes and basidiomycetes.

Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity.

BackgroundThe Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters.ResultsRepresentative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed.ConclusionIn the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members.

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganese-hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11–EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.

Genome-wide analysis of plant metal transporters, with an emphasis on poplar.

The specific transport of metal ions, mediated by membrane-localized metal transporters, is of fundamental importance in all eukaryotes. Genome-wide analysis of metal transporters was undertaken, making use of whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, as well as of the yeast Saccharomyces cerevisiae. A repertoire of 430 metal transporters was found in total across eight photosynthetic plants, as well as in S. cerevisiae. Seventy-two full-length metal transporter genes were identified in the Populus genome alone, which is the largest number of metal transporters genes identified in any single species to date. Diversification of some transporter family gene clusters appears to have occurred in a lineage-specific manner. Expression analysis of Populus metal transporters indicates that some family members show tissue-specific transcript abundance. Taken together, the data provide a picture into the diversification of these important gene families.

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii☆

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse

Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5meC levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. DOI: http://dx.doi.org/10.7554/eLife.06205.001

The anti-HIV cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes

A novel protein family homologous to the sugar‐binding antiviral protein cyanovirin‐N (CVN) is described. CVN, an 11‐kDa protein that, by binding to the high‐mannose moiety of certain viral surface glycoproteins, blocks virus entry into target cells, has thus far been identified only in the cyanobacterium Nostoc ellipsosporum. Here we show that CVN belongs to a protein family identified by analysis of transcript sequences deriving from a gene expression profiling study conducted in the truffle Tuber borchii. Members of this family (named CyanoVirin‐N Homology) are found in filamentous ascomycetes and in the fern Ceratopteris richardii. As revealed by 3D structure‐based searches, all CVNH proteins have a predicted fold that matches the so far unique fold of the cyanobacterial polypeptide. The CVNH domain is a versatile protein module. In ferns and cyanobacteria it is found in secretory proteins. In filamentous ascomycetes it is found in nonsecretory monodomain proteins as well as part of multidomain proteins bearing functionally related modules such as the peptidoglycan and chitin‐binding domain LysM. Transcript abundance data further indicate that the expression of different CVNH forms is modulated in response to nutrient availability. These findings have implications for the understanding of protein–oligosaccharide interaction in fungi and plants, and provide candidate polypeptides to be tested and exploited as antiviral agents. Proteins 2005.

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K(m)=4.7 microM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells.

Ability of Bifidobacterium breve To Grow on Different Types of Milk: Exploring the Metabolism of Milk through Genome Analysis

ABSTRACT We have investigated the occurrence of bifidobacteria in human milk samples, and we provide evidence regarding the predominance of members of the Bifidobacterium breve species in this environment. Moreover, evaluation of the growth capabilities and transcriptomic analyses of one representative isolate of this species, i.e., B. breve 4L, on different milk types were performed.