Rapid Divergence of Genome Architectures Following the Origin of an Ectomycorrhizal Symbiosis in the Genus Amanita.

Fungi are evolutionary shape shifters and adapt quickly to new environments. Ectomycorrhizal (EM) symbioses are mutualistic associations between fungi and plants and have evolved repeatedly and independently across the fungal tree of life, suggesting lineages frequently reconfigure genome content to take advantage of open ecological niches. To date analyses of genomic mechanisms facilitating EM symbioses have involved comparisons of distantly related species, but here, we use the genomes of three EM and two asymbiotic (AS) fungi from the genus Amanita as well as an AS outgroup to study genome evolution following a single origin of symbiosis. Our aim was to identify the defining features of EM genomes, but our analyses suggest no clear differentiation of genome size, gene repertoire size, or transposable element content between EM and AS species. Phylogenetic inference of gene gains and losses suggests the transition to symbiosis was dominated by the loss of plant cell wall decomposition genes, a confirmation of previous findings. However, the same dynamic defines the AS species A. inopinata, suggesting loss is not strictly associated with origin of symbiosis. Gene expansions in the common ancestor of EM Amanita were modest, but lineage specific and large gene family expansions are found in two of the three EM extant species. Even closely related EM genomes appear to share few common features. The genetic toolkit required for symbiosis appears already encoded in the genomes of saprotrophic species, and this dynamic may explain the pervasive, recurrent evolution of ectomycorrhizal associations.

Biochemical characteristics of a novel protease from the basidiomycete Amanita virgineoides.

The characterization of a novel protease from Amanita virgineoides is described. The A. virgineoides protease was purified to homogeneity using Q-Sepharose, carboxymethyl-cellulose, diethylaminoethyl-cellulose, and a gel filtration step on Superdex 75. The molecular mass of the purified protease was estimated to be 16.6 kDa. The protease was purified 32.1-fold, and its specific activity was 301.4 U/mg. The optimum pH was 4.0, and the optimum temperature was 50 °C. Kinetic constants (Km , Vmax ) were determined under the optimum reaction conditions, with Km and Vmax , being 3.74 mg/mL and 9.98 µg mL-1 Min-1 , respectively. The activity of the protease was curtailed by Cu2+ , Pb2+ , Fe3+ , Cd2+ , and Hg2+ ions but enhanced by Mg2+ , Ca2+ , and K+ ions at low concentrations. The protease activity was adversely affected by ethylene diamine tetraacetic acid, suggesting that it is a metalloprotease. Four peptide sequences were obtained from liquid chromatography-tandem mass spectrometry, including KQALSGIR, TIAMDGTEGLVR, VALTGLTVAEYFR, and AGAGSATLSMAYAGAR, which showed 86%, 64%, 60%, and 75% identity with peptides of Hypsizygus marmoreus, Dacryopinax sp. DJM-731 SS1, Trametes versicolor FP-101664 SS1, and Paxillus involutus ATCC 200175, respectively. The newly isolated protease showed good hydrolytic activity and biochemical characteristics, which expanded the knowledge of biologically active proteins and provided further insight on this poisonous fungus.

Horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal Amanita.

The genus Amanita encompasses both symbiotic, ectomycorrhizal fungi and asymbiotic litter decomposers; all species are derived from asymbiotic ancestors. Symbiotic species are no longer able to degrade plant cell walls. The carbohydrate esterases family 1 (CE1s) is a diverse group of enzymes involved in carbon metabolism, including decomposition and carbon storage. CE1 genes of the ectomycorrhizal A. muscaria appear diverged from all other fungal homologues, and more similar to CE1s of bacteria, suggesting a horizontal gene transfer (HGT) event. In order to test whether AmanitaCE1s were acquired horizontally, we built a phylogeny of CE1s collected from across the tree of life, and describe the evolution of CE1 genes among Amanita and relevant lineages of bacteria. CE1s of symbiotic Amanita were very different from CE1s of asymbiotic Amanita, and are more similar to bacterial CE1s. The protein structure of one CE1 gene of A. muscaria matched a depolymerase that degrades the carbon storage molecule poly((R)-3-hydroxybutyrate) (PHB). Asymbiotic Amanita do not carry sequence or structural homologues of these genes. The CE1s acquired through HGT may enable novel metabolisms, or play roles in signaling or defense. This is the first evidence for the horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal fungi.

Peptide macrocyclization catalyzed by a prolyl oligopeptidase involved in α-amanitin biosynthesis.

Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. Here, we show that a specific POP (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.

Ribosomally encoded cyclic peptide toxins from mushrooms.

The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

Amanita thiersii is a saprotrophic fungus expanding its range in the United States.

Although most species in the genus Amanita form ectomycorrhizal associations, a few are reported to be saprotrophs living in grassland habitats. Little is known about the ecology and distribution of these free-living Amanita species. We describe the ecology of Amanita thiersii, a species commonly collected in lawns throughout the Mississippi River Basin. Stable isotopes of carbon, transcriptomic sequences and patterns of growth on complex carbon sources provide evidence for A. thiersii as a saprotrophic species. Sporocarps of A. thiersii are less depleted in (13)C compared to published data for ectomycorrhizal fungi, supporting a saprotrophic mode of carbon acquisition in the field. Orthologs of cellulase genes known to play key roles in the decomposition of cellulose in other basidiomycetes were identified in a transcriptome of A. thiersii, establishing that this species has the genetic potential to degrade cellulose. Amanita thiersii also can use artificial cellulose or sterile grass litter as a sole carbon source. DNA sequences of three nuclear gene regions and banding patterns from four inter-simple sequence repeat markers were identical across 31 populations from throughout the known range of the species, which suggests the genetic diversity of A. thiersii populations is low. Maps of A. thiersii collections made from the 1950s until present suggest this species is experiencing a range expansion. It was reported first in 1952 in Texas and now occurs in nine states north to Illinois. These data provide an ecological context for interpreting the genome of A. thiersii, currently being sequenced at the United States Department of Energy's Joint Genome Institute.

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of Trichoderma.

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.

Colocalization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps.

Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the "MSDIN" gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.

Ectomycorrhizal fungi and their enzymes in soils: is there enough evidence for their role as facultative soil saprotrophs?

Although ectomycorrhizal (ECM) fungi are generally regarded as dependent upon the supply of carbon from their plant hosts, some recent papers have postulated a role for these fungi in the saprotrophic acquisition of carbon from soil. This theory was mainly based on the increase in enzymatic activity during periods of low photosynthate supply from tree hosts and emergence of the theory has led to a question about the overall influence of saprotrophy by ECM fungi on soil carbon turnover. However, I argue here that there is still not enough evidence to confirm this proposed function. My argument is based on inference from several lines of observation and concern over several aspects of the past studies. First, ECM fungi mainly inhabit deeper soil horizons, in which the availability of carbon compounds with positive energetic value is low. Second, the ability of ECM fungi to produce ligninolytic enzymes and cellulases is much weaker than that of saprotrophic basidiomycetes. This is most apparent in the low copy abundance of corresponding genes in the sequenced genomes of ECM species Laccaria bicolor and Amanita bisporigenes compared to the saprotrophic species Galerina marginata. I offer alternative hypotheses to explain the past observations of increased enzyme activity during starvation periods. These include, the induction of autolytic processes in ECM fungal mycelia or an attack on the host tissues to support escape from a dying root and to allow for a search for new hosts.

Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.

Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based "shotgun" proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial "cellulase" preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.

Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi.

The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas.

To obtain photoassimilates in ectomycorrhizal symbiosis, the fungus has to create a strong sink, for example, by conversion of plant-derived hexoses into fungus-specific compounds. Trehalose is present in large quantities in Amanita muscaria and may thus constitute an important carbon sink. In Amanita muscaria-poplar (Populus tremula x tremuloides) ectomycorrhizas, the transcript abundances of genes encoding key enzymes of fungal trehalose biosynthesis, namely trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP) and trehalose phosphorylase (TP), were increased. When mycorrhizas were separated into mantle and Hartig net, TPS, TPP and TP expression was specifically enhanced in Hartig net hyphae. Compared with the extraradical mycelium, TPS and TPP expression was only slightly increased in the fungal sheath, while the increase in the expression of TP was more pronounced. TPS enzyme activity was also elevated in Hartig net hyphae, displaying a direct correlation between transcript abundance and turnover rate. In accordance with enhanced gene expression and TPS activity, trehalose content was 2.7 times higher in the Hartig net. The enhanced trehalose biosynthesis at the plant-fungus interface indicates that trehalose is a relevant carbohydrate sink in symbiosis. As sugar and nitrogen supply affected gene expression only slightly, the strongly increased expression of the investigated genes in mycorrhizas is presumably developmentally regulated.

Sugar- and nitrogen-dependent regulation of an Amanita muscaria phenylalanine ammonium lyase gene.

The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.

The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation.

Genomic and cDNA clones derived from the gene (dodA) coding for DOPA dioxygenase, a key enzyme in the betalain pathway, were obtained from the basidiomycete Amanita muscaria. A cDNA library was established in the phage lambda ZapII and dodA clones were isolated using polyclonal antibodies raised against the purified enzyme. Their identity was confirmed by comparison of the deduced amino acid sequence with the sequence of several tryptic peptide fragments of DOPA dioxygenase. The gene coded for a 228-amino acid protein that showed no homology to published sequences. The coding region was interrupted by five short introns. Regulation was shown to occur at the transcriptional level; the mRNA accumulated to high levels only in the coloured cap tissue. dodA was found to be a single-copy gene in A. muscaria. To our knowledge, this is the first gene from the betalain pathway to be cloned. It encodes a type of aromatic ring-cleaving dioxygenase that has not been previously described.

Influence of copper, manganese and pH on the growth and several enzyme activities in mycorrhizal fungus Amanita muscaria.

The effects of various concentrations of copper, manganese and pH on the growth and several enzyme activities of mycorrhizal fungus Amanita muscaria were investigated. Cu (5-25 mg l-1) and lower pH (3.0-4.0) strongly inhibited the mycelial growth (dry weight), however, the protein content was not affected evidently. Some enzyme activities were lower as the Cu and Mn concentrations were higher and other enzymes had the maximum values at the specified concentration. The activities of the following enzymes were significantly correlated with the fungal growth after the treatment with Cu: G6PDH, MTLDH and trehalase, and with Mn: G6PDH, MTLDH and alpha-mannosidase respectively. Measurement of these enzyme activities might provide a useful biochemical criterion for the evaluation of the fungitoxicity of soil contaminated by copper or manganese.

alpha-Amanitin-resistant RNA polymerase II from carpophores of Amanita species accumulating amatoxins.

DNA-dependent RNA polymerase activities have been partially purified from carpophores of the amanitin (amatoxin)-accumulating species Amanita hygroscopica and Amanita suballiacea and the non-accumulating species Amanita brunnescens and Amanita alliacea. RNA polymerase II activities purified by ion-exchange chromatography were characterized with respect to ionic strength, template, and divalent metal ion requirements and sensitivities to inhibition by alpha-amanitin. The Ki values of alpha-amanitin for RNA polymerase II activities were: 2.0 . 10(-3) M for A. hygroscopica; 3.3 . 10(-3) M for A. suballiacea; 9.8 . 10(-6) M for A. brunnescens; 10.0 . 10(-6) M for A. alliacea. Further purification with DNA affinity chromatography of activities from A. suballiacea and A. brunnescens did not alter the apparent dissociation constants of alpha-amanitin from either enzyme. The correlation between amanitin sensitivity of RNA polymerase II and the quantity of amatoxins found in carpophores suggests these peptides may play a role in regulating transcription of messenger RNA during carpophore development.

The insensitivity of mushroom nuclear RNA polymerase activity to inhibition by amatoxins.

Nuclear fractions isolated from Amanita phalloides, Amanita muscaria and Agaricus bisporus were subjected to in vitro RNA synthesis assays in the presence of various concentrations of amatoxins. The mushroom nuclei were highly insensitive to inhibition by amatoxin when compared to assays of nuclear fractions isolated from the Oömycete fungus, Achlya ambisexualis and from rabbit brain.