Soil microbial functional gene dataset associated with Agathis australis.

Agathis australis (New Zealand kauri) is a significant and iconic native tree of Aotearoa New Zealand. Currently, Phytophthora agathidicida that causes kauri-dieback disease is killing kauri trees. Only 1% of the New Zealand virgin kauri forest remains [1,2]. Recent studies revealed that many soil-borne microorganisms had been found to systemically boost the defensive capacity of the trees by providing competition to pathogens for nutrient intake, thus preventing pathogen colonization and modulating plant immunity [3,4]. In addition, the root microbiome consists of an entire complex rhizosphere-associated microbes with their genetic elements and interactions that have influenced plant health. To date, very few studies have been conducted to investigate the microorganisms in the kauri soil and possible environmental drivers. To characterize the functional gene profile in relation to soil microbial diversity of the kauri trees at Auckland Botanic Gardens (ABG), Auckland, New Zealand the GeoChip 5.0 M (Glomics Inc. USA), a microarray-based metagenomics tool, was used. GeoChip 5.0 M comprises of 162,000 probes from 365,000 target genes (coding DNA sequence - CDS), which covers all taxonomic groups (archaea, bacteria, fungi, protists, algae, and viruses) [5]. The ABG has kauri trees that are approximately 20 years old, located in three distinct man-made environments: Native Forest, Kauri Grove, and Rose Garden. We selected two trees from the Native Forest and two from the Kauri Grove for our experiment. Soil samples were collected from the four cardinal points of each tree, at 10 cm depth. Pooled environmental DNA was sent to Glomics (USA) and the data were preprocessed using GeoChip data analysis pipeline described in http://www.ou.edu/ieg/tools/data-analysispipeline.html. Based on the GeoChip data generated from the soil samples, we have detected a total of 946 genes, 4342 taxa, 102 phyla, and 995 genera. The data presented here provide an overview of functional genes associated with kauri soil, which can serve as baseline for other kauri soil microbiome analysis at forest-scale studies. The raw data has been uploaded to Mendeley Data https://doi.org/10.17632/T22NNN385K.1.

Addressing the diversity of Xylodon raduloides complex through integrative taxonomy.

In this study, the taxonomic diversity of the Xylodon raduloides species complex (Hymenochaetales, Basidiomycota) is examined. Specimens were studied using an integrative taxonomic approach that includes molecular phylogenetic and morphological analyses, and environmental niche comparisons. Four different species were found inside the Xylodon raduloides complex, with a biogeographic distribution pattern bound by geographic regions: Europe, North America, Patagonia, and Australia-New Zealand. Molecular, morphological, and environmental evidences delimit two lineages within this complex: a Northern Hemisphere clade with longer basidiospores and wider ranges in temperature and precipitation tolerance, and a Southern Hemisphere clade with smaller and more spherical basidiospores, and an isothermal and more humid climate preference. The integrative taxonomic approach used in this study demonstrates congruence between data sets and shows how morphological and environmental characteristics contribute to the differentiation of fungal species complexes. By combining various sources of taxonomic information, three new species are described: Xylodon laurentianus, X. novozelandicus, and X. patagonicus.

Competing sexual and asexual generic names in Pucciniomycotina and Ustilaginomycotina (Basidiomycota) and recommendations for use.

With the change to one scientific name for pleomorphic fungi, generic names typified by sexual and asexual morphs have been evaluated to recommend which name to use when two names represent the same genus and thus compete for use. In this paper, generic names in Pucciniomycotina and Ustilaginomycotina are evaluated based on their type species to determine which names are synonyms. Twenty-one sets of sexually and asexually typified names in Pucciniomycotina and eight sets in Ustilaginomycotina were determined to be congeneric and compete for use. Recommendations are made as to which generic name to use. In most cases the principle of priority is followed. However, eight generic names in the Pucciniomycotina, and none in Ustilaginomycotina, are recommended for protection: Classicula over Naiadella, Gymnosporangium over Roestelia, Helicobasidium over Thanatophytum and Tuberculina, Melampsorella over Peridermium, Milesina over Milesia, Phragmidium over Aregma, Sporobolomyces over Blastoderma and Rhodomyces, and Uromyces over Uredo. In addition, eight new combinations are made: Blastospora juruensis, B. subneurophyla, Cronartium bethelii, C. kurilense, C. sahoanum, C. yamabense, Milesina polypodii, and Prospodium crusculum combs. nov.

Structural character evolution in Pucciniomycotina: mitosis, septa, and hyphal branch initiation in two Helicogloea species.

Early diverging taxa of Ascomycota and Basidiomycota share similarities in subcellular characters of the spindle pole body (SPB), nuclear division, and septal pore apparatus, but our understanding of character evolution is incomplete because of the limited number of structural studies within the earliest diverging subphyla of Dikarya, Taphrinomycotina and Pucciniomycotina. Two species of Helicogloea (Atractiellomycetes) were analyzed for these characters and provide data on SPB and nuclear division for an additional class of Pucciniomycotina. A detailed analysis of septal pore apparatus for the Helicogloea species permits comparisons with those of other Pucciniomycotina and Ascomycota. The endogenous origin of hyphal branches is shown to occur in a third class of Pucciniomycotina. The full set of characters supports a close relationship between Atractiellomycetes and Pucciniomycetes.

The arbuscular mycorrhizal fungi colonising roots and root nodules of New Zealand kauri Agathis australis.

As the only endemic member in New Zealand of the ancient conifer family, Araucariaceae, Agathis australis is an ideal species to study putatively long-evolved mycorrhizal symbioses. However, little is known about A. australis root and nodular arbuscular mycorrhizal fungi (AMF), and how mycorrhizal colonisation occurs. We used light, scanning and transmission electron microscopy to characterise colonisation, and 454-sequencing to identify the AMF associated with A. australis roots and nodules. We interpreted the results in terms of the edaphic characteristics of the A. australis-influenced ecosystem. Representatives of five families of Glomeromycota were identified via high-throughput pyrosequencing. Imaging studies showed that there is abundant, but not ubiquitous, colonisation of nodules, which suggests that nodules are mostly colonised by horizontal transmission. Roots were also found to harbour AMF. This study is the first to demonstrate the multiple Glomeromycota lineages associated with A. australis including some that may not have been previously detected.

The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction.

Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8 Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.

Saprotrophic fungal mycorrhizal symbionts in achlorophyllous orchids: finding treasures among the 'molecular scraps'?

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific of each mycoheterotrophic species and (2) mycorrhizal on surrounding green plants, which are the ultimate carbon source of the entire system. Here we review recent works revealing that some mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as 'molecular scraps', since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.

Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.

Deletion of the acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic beta-cell dysfunction, diabetes, and the metabolic syndrome.

Conservation of cytoplasmic organization in the cystidia of Suillus species.

Cystidia of Suillus americanus and S. granulatus (Boletales) were examined cytochemically and ultrastructurally with cells prepared by freeze substitution. We present the first study showing ultrastructural details and cytological functions of the cystidium to be conserved in two closely related species. The results are presented for inclusion in the AFTOL Structural and Biochemical Database to aid in the application of morphological characters to phylogenetic studies. The cystidia of these Suillus species appear to be united by a series of conserved characters, including specialized secretion mechanisms, smooth tubular endoplasmic reticulum and abundant free ribosomes. The conservation of these subcellular traits among members of this genus suggests that ultrastructural details of cystidia may provide a suite of phylogenetically informative characters. Inclusion of such characters in phylogenetic analyses might resolve or provide support for monophyletic groups at the level of family or genus.

The mushroom family Psathyrellaceae: evidence for large-scale polyphyly of the genus Psathyrella.

Psathyrella is the archetypal little brown mushroom genus with few easily discernable characters causing it to be considered a "clean-up" genus for other small brown-spored saprotrophic species found worldwide. While molecular studies have demonstrated that mushroom genera based on homoplastic morphological characters are artificial, the degree of phylogenetic heterogeneity contained within Psathyrella and Psathyrellaceae has never been appropriately addressed. For this study, 132 ribosomal sequences from approximately one-tenth of the known Psathyrella species worldwide, including representatives of most subgeneric subdivisions, and three closely related coprinoid genera (Parasola, Coprinopsis, Coprinellus) were evaluated using multiple phylogenetic methods, including likelihood, with Agaricaceae as the outgroup. Our results indicated that Psathyrella was polyphyletic. Conservatively, the genus can be separated into 11 clades of which five can be raised to generic status. Most species of Psathyrella, including its type species P. gracilis, formed a large clade with Coprinellus, which appeared to be derived from within Psathyrella. Generic limits of Parasola, Lacrymaria, and Coprinopsis should be reevaluated. Several taxa previously synonymized based on morphological features were phylogenetically distinct. Morphological features traditionally used to subdivide Psathyrella appeared to be mostly convergent (homoplasious) when traced upon the resulting phylogenies, although several had high RI values. These results were interpreted in light of the two major taxonomic treatments of Psathyrella and revealed substantial inconsistencies between the molecular- and morphology-derived inferences of relationships.

Septal pore apparatus and nuclear division of Auriscalpium vulgare.

Ultrastructure of the septal pore apparatus and nuclear division of Auriscalpium vulgare (Russulales) was examined with freeze substitution and is presented for inclusion in the AFTOL Structural and Biochemical Database (http://aftol.umn.edu). Previously unreported septal characters for the Russulales (Agaricomycotina) were observed: Septa of the hymenophore had bell-shaped perforated septal pore caps that may extend along the septum and a zone of organelle exclusion surrounded the septal pore apparatus. Metaphase I of meiosis and metaphase of mitosis were similar. Globular spindle pole bodies with electron-opaque inclusions were set within polar fenestrae of the nuclear envelope. The nuclear envelope was mostly intact with occasional gaps. Fragments of endoplasmic reticulum were present near the spindle pole bodies but did not form a polar cap. Structural characters may distinguish one or more clades of the Agaricomycotina and provide additional signal in phylogenetic analyses.

Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.

The esterification of alcohols such as sterols, diacylglycerols, and monoacylglycerols with fatty acids represents the formation of both storage and cytoprotective molecules. Conversely, the overproduction of these molecules is associated with several disease pathologies, including atherosclerosis and obesity. The human acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 gene superfamily comprises seven members, four of which have been previously implicated in the synthesis of di- or triacylglycerol. The remaining 3 members comprise an X-linked locus and have not been characterized. We describe here the expression of DGAT2 and the three X-linked genes in Saccharomyces cerevisiae strains virtually devoid of neutral lipids. All four gene products mediate the synthesis of triacylglycerol; however, two of the X-linked genes act as acyl-CoA wax alcohol acyltransferases (AWAT 1 and 2) that predominantly esterify long chain (wax) alcohols with acyl-CoA-derived fatty acids to produce wax esters. AWAT1 and AWAT2 have very distinct substrate preferences in terms of alcohol chain length and fatty acyl saturation. The enzymes are expressed in many human tissues but predominate in skin. In situ hybridizations demonstrate a differentiation-specific expression pattern within the human sebaceous gland for the two AWAT genes, consistent with a significant role in the composition of sebum.

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution.

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.

Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits.

Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.