Traceability of Marketable Japanese Shoro in New Zealand: Using Multiplex PCR To Exploit Phylogeographic Variation among Taxa in the Rhizopogon Subgenus Roseoli

Rhizopogon roseolus Corda (synonym Rhizopogon rubescens Tul.), an economically important edible mushroom associated with the Pinaceae (mostly Pinus sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of R. roseolus varies with the place of origin. R. roseolus strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of R. roseolus and other closely related fungi belonging to Rhizopogon subgenus Roseoli to determine the genetic basis for phenotypic differences among R. roseolus isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within Rhizopogon subgenus Roseoli. Collections from the United States and Europe grouped into four distinct clades. Rhizopogon roseolus isolates found in New Zealand were closely related to those from the United States, likely due to introduction of Pinus radiata from its native California in the United States. In contrast, Japanese R. roseolus isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand R. roseolus isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand R. roseolus isolates to track the establishment of ectomycorrhiza on P. radiata seedlings inoculated with commercially valuable R. roseolus. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on P. radiata in the Southern Hemisphere.Since the latter half of the 19th century, Northern Hemisphere species of exotic trees, in particular Pinus radiata, have been planted in the Southern Hemisphere (e.g., Chile, Argentina, South Africa, Australia, and New Zealand). Indeed, over 1,000,000 ha of exotic trees have been established in New Zealand alone (32, 9). Over 200 nonnative basidiomycete and ascomycete ectomycorrhizal (ECM) fungal species are associated with Pinus or Eucalyptus plantations in the Southern Hemisphere from the introduction of plants with intact root systems (46).The genus Rhizopogon Fries (Basidiomycota, Boletales) contains more than 100 species of hypogeous fungi (24), which form ECM associations mostly with members of the Pinaceae (44). The greatest diversity of Rhizopogon can be found in the coniferous forests of the Pacific northwestern United States (40, 30), although a number of species are known to occur in Europe (40) and Asia (3). At least four introduced species of Rhizopogon have also been reported in New Zealand, including R. clelandii Cunn., R. luteolus Fr., R. roseolus (7), and R. vinicolor A. H. Smith (5). At least two species, R. luteolus and R. roseolus, have been associated with P. radiata seedlings in forest nurseries in the North Island of New Zealand (6).In Japan, R. roseolus is known locally as shoro. Shoro is considered a delicacy (20), and its production is dependent on the collection of the carpophores in the field. In fact, only 200 years ago, shoro was the fourth most commonly consumed mushroom in Japan (34). However, the number of natural shoro has declined in the second half of the 20th century (15), resulting in its cultivation in forestry plantations since the late 1980s (50). To fulfill Japanese demand, plantations of P. radiata artificially inoculated with R. roseolus have been established in New Zealand since 1999 using spores from fruiting bodies collected locally (47). Three of the four plantations have subsequently produced fruiting bodies, but the crop has been deemed unsuitable due to consumer sensitivity in Japan to the origin of the products and doubts surrounding the authenticity of the fruiting bodies as Japanese shoro. The quality and market price of other edible fungi differ not only with species but also with their origin. In Japan, domestic Tricholoma matsutake is considered the premium source of matsutake, traded at $500/kg, while South Korean matsutake and those from China are considered less valuable ($250/kg and $100/kg, respectively) (31). Highly prized edible fungi have also been found to be contaminated with less valuable species. Tuber rufum Pico is a truffle species that is found alongside other valuable species in countries where truffles are commercially important. However, it is considered to be a poorly flavored species with no marketable value and is deemed a “contaminant” in truffières (4).Unfortunately, the taxonomy of the genus Rhizopogon is surrounded by many unresolved issues, which makes differentiation of shoro-like fungi found in different geographical locations difficult. In 1966, Smith and Zeller (40) completed the first taxonomic study of the genus Rhizopogon, dividing it into two subgenera, Rhizopogonella (subsequently transferred to Alpova [45]) and Rhizopogon. The subgenus Rhizopogon was further divided into four sections based on differences in the host plant, sporocarp morphology, and color of the peridium. All species associated with Pinus spp. were classified in Rhizopogon section Rhizopogon. Rhizopogon roseolus and R. rubescens were placed in subsection Angustispori, stirps Rubescens. A third species, Rhizopogon vulgaris Vittad., was placed in subsection Angustispori, stirps Vulgaris, due to differences in its spore morphology.Similarities between species in stirps Rubescens and stirps Vulgaris were recorded, with stirps Vulgaris considered a continuation of stirps Rubescens into the narrow-spore species. However, classification was based only on examinations of North American collections even though these species were originally described in Europe in the 19th century (40). Taxonomic reexamination of Rhizopogon using phylogenetic analyses of internal transcribed spacer (ITS) DNA sequences showed that Rhizopogon burlinghamii, R. roseolus, and R. vulgaris formed Rhizopogon section Rhizopogon clade C, separate from the other species sampled from section Rhizopogon (clades A and B). Their ITS sequences lacked insertions and deletions that are diagnostic of other section Rhizopogon clades. Rhizopogon roseolus and R. vulgaris were placed together under Rhizopogon subgenus Roseoli (13). Unfortunately, collections classified as R. rubescens were not included in the phylogenetic study. More recently, species concepts in the R. roseolus species group were examined by Martín and García (25). ITS sequence analyses separated the collections into five possible phylogenetic species.The continued taxonomic instability of Rhizopogon subgenus Roseoli has created ongoing confusion, and the correct species names are still not clear. In Japan, until now, shoro has been referred to as R. rubescens; however, R. rubescens is used widely as a synonym for R. roseolus. In this study, R. roseolus will be used to describe all collections unless specifically stated, as this taxonomic name appears to have precedence in previous phylogenetic studies of Rhizopogon (13, 19, 40) and since Mycobank (http://www.MycoBank.org/) considers R. rubescens to be a synonym of R. roseolus.Molecular diagnostic tools capable of distinguishing genetic differences in ECM fungi have been developed, allowing the differentiation of commercially important species from contaminants or similar species of less economic value. There are many studies where PCR primers designed for the amplification of the ITS region have been used to identify basidiomycetes (12, 18). Species-specific primers were created to identify and differentiate marketable boletes (28), to detect black truffle species (37), and to distinguish Asiatic black truffles from Tuber melanosporum in commercialized products (22). A multiplex PCR has also been developed to simultaneously detect different white truffle species and one of the most aggressive contaminant fungi for monitoring the persistence of a selected truffle in inoculated seedlings (1).Due to the sensitivity of consumers to the origins of shoro and the existing taxonomic complexity of the genus Rhizopogon, morphological and molecular methods were used to establish the diversity and genetic structure of Rhizopogon subgenus Roseoli. Phylogenetic relationships between shoro-like species (originally classified as R. vulgaris, R. rubescens, and R. roseolus) from different geographical locations were investigated to verify previously observed differences between shoro grown in Japan and New Zealand. A multiplex PCR was then developed for the rapid identification of ECMs and fruiting bodies grown from Japanese shoro in New Zealand to track the commercial production of this economically valuable edible fungus.     

Formation and function of a new pollen aperture pattern in angiosperms: The proximal sulcus of Tillandsia leiboldiana (Bromeliaceae)

Pollen grains are generally surrounded by an extremely resistant wall interrupted in places by apertures that play a key role in reproduction; pollen tube growth is initiated at these sites. The shift from a proximal to distal aperture location is a striking innovation in seed plant reproduction. Reversals to proximal aperture position have only very rarely been described in angiosperms. The genus Tillandsia belongs to the Bromeliaceae family, and its aperture pattern has been described as distal monosulcate, the most widespread aperture patterns recorded in monocots and basal angiosperms. Here we report developmental and functional elements to demonstrate that the sulcate aperture in Tillandsia leiboldiana is not distal as previously described but proximal. Postmeitotic tetrad observation indicates unambiguously the proximal position of the sulcus, and in vitro germination of pollen grains confirms that the aperture is functional. This is the first report of a sulcate proximal aperture with proximal germination. The observation of microsporogenesis reveals specific features in the patterns of callose thickenings in postmeiotic tetrads.     

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.Later steps of carotenoid biosynthesis involve the formation of diverse cyclic carotenoids. For example, β-carotene, the vitamin A precursor, is synthesized de novo by photosynthetic organisms, limited nonphototrophic bacteria and fungi, and also by aphids (Moran and Jarvik, 2010) according to a multistep pathway that ends with the cyclization of lycopene by lycopene β-cyclase (LCY-B). Similarly, in pepper (Capsicum annuum) chromoplasts, antheraxanthin and violaxanthin are converted into the κ-cyclic carotenoids capsanthin and capsorubin, respectively, by capsanthin-capsorubin synthase (CCS). In both cases, the proposed mechanism involves a concerted protic attack and stabilization of a transient carbocation without any net redox change (Camara, 1980; Bouvier et al., 1994; Britton, 1998). Several cDNAs for LCY-B have been cloned from bacteria (Misawa et al., 1990; Cunningham et al., 1994; Armstrong, 1997; Cunningham and Gantt, 2001), fungi (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001), and plants (Hugueney et al., 1995; Ronen et al., 2000) using functional complementation. Information available from primary structures suggest that the cyclization of lycopene is catalyzed by holomeric proteins in photosynthetic organisms (Cunningham et al., 1994; Maresca et al., 2007), by holomeric (Misawa et al., 1990) or heteromeric (Krubasik and Sandmann, 2000; Viveiros et al., 2000) proteins in nonphotosynthetic bacteria, and by holomeric, bifunctional proteins in fungi that combine the activities of phytoene synthase and lycopene cyclase (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001). This structural diversity of LCY-Bs coupled to a lack of significant amino acid sequence identity between the lycopene cyclases from bacteria, fungi, and plants hinder our understanding of the catalytic mechanism of LCY-Bs and CCS. In addition, the N terminus of plant LCY-B and CCS contains an amino sequence motif characteristic of a polypeptide predicted to adopt a Rossmann fold (Rossmann et al., 1974) and suggests the binding of an as yet unknown dinucleotide prosthetic ligand. It has been shown using recombinant bacterial enzyme that the cyclization of lycopene into β-carotene strictly requires NADPH but proceeds without any net redox change (Schnurr et al., 1996; Hornero-Mendez and Britton, 2002). Under the same conditions, FAD alone could not sustain bacterial LCY-B activity (Schnurr et al., 1996). Much less is known about the dinucleotide requirements of plant carotenoid cyclases, which are highly conserved within plants but are extremely divergent in nonplant organisms. Previously, a crucial acidic domain for lycopene cyclase activity was identified using an affinity-labeling strategy followed by site-directed mutagenesis (Bouvier et al., 1997) in the absence of any crystal structures. This so-called 293-FLEET-297 motif of LCY-B and CCS contained two tandem Glu-295-Glu-296 residues that were essential for LCY-B- and κ-cyclase activities (Bouvier et al., 1997). However, it still remains unclear how the protic mechanism is compatible with the requirement of dinucleotide cofactors.To further explore the mechanism of plant carotenoid cyclases, we first choose pepper CCS as a prototypic enzyme because it displays a strong identity (52%) to pepper LCY-B, and we have shown previously that CCS could also catalyze the cyclization of lycopene into β-carotene (up to 25% of activity compared with LCY-B; Hugueney et al., 1995). Herein, we have shown that monomeric CCS purified to homogeneity from plant chromoplasts or recombinant CCS purified from Escherichia coli-transformed cells are typical flavoproteins containing one noncovalently bound FAD. We also observed that CCS-bound FAD is required for enzyme activity in the presence of NADPH, which functions as a reductant of FAD. During this process, no hydrogen is transferred to β-carotene or κ-cyclic carotenoids. In addition to this cofactor requirement, we also show from extensive site-directed mutagenesis using pepper CCS and LCY-B and Erwinia herbicola LCY-B (Mialoundama, 2009) that Glu-295 of pepper CCS and LCY-B plays a key role in the formation of β-carotene and κ-cyclic carotenoids, and we demonstrate that a similar role is played in structurally divergent bacterial LCY-Bs by Glu-196. These characteristics suggest that plant CCS and LCY-Bs are mechanistically similar to non-metal-assisted terpene cyclases, such as squalene:hopene cyclase and oxidosqualene cyclase, and additionally represent a new subfamily of flavoproteins like isopentenyl diphosphate isomerase type II, which catalyze carotenoid cyclization without any net redox modification of the substrate.     

B cells from rheumatoid arthritis patients show important alterations in the expression of CD86 and FcγRIIb,which are modulated by anti-tumor necrosis factor therapy

Introduction  

Several molecules help preserve peripheral B cell tolerance, but when altered, they may predispose to autoimmunity. This work studied the expression of the costimulatory molecule CD86 and the inhibitory receptor for IgG immune complexes FcγRIIb (CD32b), on B cells from rheumatoid arthritis (RA) patients, and the influence of anti-tumor necrosis factor (TNF) therapy.     

Tyrosine-Phosphorylated Caveolin-1 Blocks Bacterial Uptake by Inducing Vav2-RhoA-Mediated Cytoskeletal Rearrangements

Certain bacterial adhesins appear to promote a pathogen''s extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P⁺GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.     

Correction to: Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs

Corals house a variety of microorganisms which they depend on for their survival, including endosymbiotic dinoflagellates (Symbiodiniaceae) and bacteria. While cnidarian–microorganism interactions are widely studied, Symbiodiniaceae–bacteria interactions are only just beginning to receive attention. Here, we describe the localization and composition of the bacterial communities associated with cultures of 11 Symbiodiniaceae strains from nine species and six genera. Three-dimensional confocal laser scanning and electron microscopy revealed bacteria are present inside the Symbiodiniaceae cells as well as closely associated with their external cell surface. Bacterial pure cultures and 16S rRNA gene metabarcoding from Symbiodiniaceae cultures highlighted distinct and highly diverse bacterial communities occur intracellularly, closely associated with the Symbiodiniaceae outer cell surface and loosely associated (i.e., in the surrounding culture media). The intracellular bacteria are highly conserved across Symbiodiniaceae species, suggesting they may be involved in Symbiodiniaceae physiology. Our findings provide unique new insights into the biology of Symbiodiniaceae.Subject terms: Symbiosis, Microbiome, Marine microbiology     

Activity-guided isolation of antiplasmodial dihydrochalcones and flavanones from Piper hostmannianum var. berbicense

The bioassay-guided purification of an n-hexane extract from the leaves of Piper hostmannianum var. berbicense led to the isolation of four monoterpene or prenyl-substituted dihydrochalcones (1a, 1b, 2, 3) as well as the known compounds 2',6'-dihydroxy-4'-methoxydihydrochalcone (4), linderatone (5), strobopinin (6), adunctin E (7) and (-)-methyllinderatin (8). Their structures were established on the basis of NMR and X-ray analysis. (-)-Methyllinderatin, linderatone and 2',6'-dihydroxy-4'-methoxydihydrochalcone exhibited the most potent antiplasmodial activity with IC50 values of 5.64, 10.33 and 12.69 microM, respectively against both chloroquine-sensitive and resistant strains of Plasmodium falciparum (F32,FcB1). The activity of (-)-methyllinderatin was confirmed in vivo against Plasmodium vinckei petteri in mice (80% of reduction of parasitemia) at a dose of 20 mg/kg/day.