Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi.

BACKGROUND: Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy. RESULTS: In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi. CONCLUSIONS: In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

The genome of Lyophyllum shimeji provides insight into the initial evolution of ectomycorrhizal fungal genomes.

Mycorrhizae are one of the most fundamental symbioses between plants and fungi, with ectomycorrhizae being the most widespread in boreal forest ecosystems. Ectomycorrhizal fungi are hypothesized to have evolved convergently from saprotrophic ancestors in several fungal clades, especially members of the subdivision Agaricomycotina. Studies on fungal genomes have identified several typical characteristics of mycorrhizal fungi, such as genome size expansion and decreases in plant cell-wall degrading enzymes (PCWDEs). However, genomic changes concerning the evolutionary transition to the ectomycorrhizal lifestyle are largely unknown. In this study, we sequenced the genome of Lyophyllum shimeji, an ectomycorrhizal fungus that is phylogenetically related to saprotrophic species and retains some saprotroph-like traits. We found that the genome of Ly. shimeji strain AT787 lacks both incremental increases in genome size and reduced numbers of PCWDEs. Our findings suggest that the previously reported common genomic traits of mycorrhizal fungi are not essential for the ectomycorrhizal lifestyle, but are a result of abolishing saprotrophic activity. Since Ly. shimeji is commercially consumed as an edible mushroom, the newly available genomic information may also impact research designed to enhance the cultivation of this mushroom.

The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation.

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

Taxonomic revision of Termitomyces species found in Ryukyu Archipelago, Japan, based on phylogenetic analyses with three loci.

Fungi in the genus Termitomyces are external symbionts of fungus-growing termites. The three rhizogenic Termitomyces species T. eurrhizus, T. clypeatus, and T. intermedius, and one species similar to T. microcarpus that lacks pseudorrhiza, have been reported from Ryukyu Archipelago, Japan. In contrast, only two genetic groups (types A and B) of Termitomyces vegetative mycelia have been detected in nests of the fungus-growing termite Odontotermes formosanus. In this study, we investigated the relationships between the mycelial genetic groups and the basidiomata of Termitomyces samples from the Ryukyu Archipelago. We found that all the basidioma specimens and the type B mycelia formed one clade that we identified as T. intermedius. Another clade consisted of the type A mycelia, which showed similarity to T. microcarpus, was identified as T. fragilis. Our results indicate that the Japanese T. eurrhizus and T. clypeatus specimens should re-named as T. intermedius.

Expression and genome-wide analysis of the xylogen-type gene family.

In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (ß-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.

[Three cases of methotrexate-associated lymphoproliferative disorder (MTX-LPD)].

Methotrexate (MTX) has been increasingly administered to patients with rheumatoid arthritis (RA), resulting in methotrexate-associated lymphoproliferative disorder (MTX-LPD) in patients. We reported three case of rheumatoid arthritis (RA) undergoing methotrexate (MTX) therapy who developed MTX-LPD. A 72-year-old woman treated with MTX since December 1997 (total dose 3684 mg) presented with swelling of the right tonsil in October 2006, and diffuse large B-cell lymphoma was diagnosed by tonsil biopsy and positive EBER1. When MTX therapy was interrupted, the tonsil was shrank and chemotherapy was not necessary. She followed a good clinical course for 12 months. Two other patients treated with MTX for RA for several years presented with enlarged neck lymph nodes and were diagnosed with MTX-LPD. Neck lymph nodes shrank upon MTX withdrawal in several weeks. There have been no signs of recurrence in these cases and they followed a good clinical course. The oncogenic potential of MTX and RA is reviewed.

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis.

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi.

Chemical Synthesis of the Epimeric (23R)- and (23S)-Fluoro Derivatives of Bile Acids via Horner-Wadsworth-Emmons Reaction.

A method for the synthesis of two (23R)- and (23S)-epimeric pairs of 23-fluoro-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid and 23-fluoro-3α,7α-dihydroxy-5ß-cholan-24-oic acid is described. The key intermediates, 23,24-dinor-22-aldehyde peracetates were prepared from cholic and chenodeoxycholic acids via the 24-nor-22-ene, 24-nor-22ξ,23-epoxy, and 23,24-dinor-22-aldehyde derivatives. The Horner-Wadsworth-Emmons reaction of the 23,24-dinor-22-aldehydes using triethyl 2-fluoro-2-phosphonoacetate in the presence of LiCl and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and subsequent hydrogenation of the resulting 23ξ-fluoro-22-ene ethyl esters, followed by hydrolysis, gave a mixture of the epimeric (23R)- and (23S)-fluorinated bile acids which were resolved efficiently by preparative RP-HPLC. The stereochemical configuration of the fluorine atom at C-23 in the newly synthesized compounds was confirmed directly by the X-ray crystallographic data. The (1)H and (13)C NMR spectral differences between the (23R)- and (23S)-epimers were also discussed.

A Phalaenopsis variety with floral organs showing C class homeotic transformation and its revertant may enable Phalaenopsis as a potential molecular genetic material.

The Orchidaceae is one of the most famous garden plants, and improvement of the orchid is very important in horticulture field. However, molecular information is largely unknown. We found a Phalaenopsis variety harboring floral organs showing C class homeotic change. Column is composed of the anthers with the receptive stigmatic surface just underneath them in wild type. However the C class variety produced column with sepal or petal like structure at the abaxial side. This is the typical abnormality as C class mutants in plants. Further, wild type looking revertant was found from the meristem tissue cultured population. This result strongly indicates the existence of active transposable element in Phalaenopsis genome. This transposon may enable Phalaenopsis as a good material for molecular genetic analysis in Orchidaceae.