Analysis of dinoflagellate mitochondrial protein sorting signals indicates a highly stable protein targeting system across eukaryotic diversity

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.     

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Colonization of Corn, Zea mays, by the Entomopathogenic Fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

Phragmidium satoanum,a new rust pathogen of Rosa hirtula in Japan

The causal fungus of a rust disease of Rosa hirtula, endemic to mountainous areas of Fuji-Hakone-Izu National Park, Japan, was thought to be a common species Phragmidium rosae-multiflorae. Continued field observations, morphological examination, and experimental inoculations proved that the fungus produced laterally three-angled aeciospores and urediniospores together with multi-cellular teliospores on the same R. hirtula trees. These morphological features were different from those of P. rosae-multiflorae. The fungus parasitized only R. hirtula. Experimental inoculations and field observations did not prove that R. banksiae, R. laevigata, and R. multiflora supported infection and sporulation of the fungus. Under the field observations, R. multiflora, the most common host of P. rosae-multiflorae, was not proven to harbor the R. hirtula fungus. Therefore, the fungus was concluded to be a species distinct from P. rosae-multiflorae; and a new name, P. satoanum, was proposed for it.     

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.     

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: Is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in Arabidopsis thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signaling in the plant.Key words: mycorrhiza, ectomycorrhiza, lateral root, auxin, volatiles, ethylene, jasmonic acidInteractions of plant roots with symbiotic, ectomycorrhizal soil fungi lead to lateral root (LR) stimulation during the very early interaction phase.¹ This LR stimulation has recently been shown to be independent of root colonization and to occur as well in non-mycorrhizal plants, such as Arabidopsis suggesting that fungal signals have a broad perception spectrum.^1,2 However, little is known about the type of signals exchanged between fungi and their plant partners during this early interaction phase. Several studies have proposed a role for the phytohormone auxin produced and secreted by ECM fungi as the signalling molecule during ECM fungus/plant signaling.^2–7 Recently we studied changes in auxin response and auxin transport in poplar and Arabidopsis thaliana roots during contact with the ECM fungus Laccaria bicolor.¹ We demonstrated that the presence of the fungus enhances the auxin response and distribution at the root apex and that this, as well as LR stimulation, is reliant on polar auxin transport through AtPIN2 and probably through PtPIN9 in poplar. Here, using Arabidopsis thaliana, whose LR stimulation by Laccaria bicolor has been demonstrated, we propose that not yet identified fungal volatiles may regulate auxin homeostasis in the plant, questioning the contribution of the auxin released by the fungus on the induction of LR.     

Haradamyces foliicola anam. gen. et sp. nov., a cause of zonate leaf blight disease in Cornus florida in Japan

A fungus causing zonate leaf blight diseases in various evergreen and deciduous woody plant species in Japan was characterized by a discoid multicellular propagule arising from a hyaline sclerotium-like structure in the leaf tissue and dark-coloured microconidia produced enteroblastically from the terminal cells on the surface of the discoid propagules. Myrioconium-like microconidiophores also producing microconidia were occasionally produced in culture. No teleomorphic characteristics were observed on the fungus. Molecular analysis based on the partial nu-rDNA sequence data revealed that the fungus was phylogenetically related to the Sclerotiniaceae, Leotiomycetes, and Ascomycota. Because the morphology and sequence data of this fungus does not coincide with those of any known anamorphic fungi, Haradamyces foliicola is proposed here as a new anamorphic genus and species for this fungus.     

In Planta Regulation of Extension of an Endophytic Fungus and Maintenance of High Metabolic Rates in Its Mycelium in the Absence of Apical Extension

The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfected L. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.     

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10^® Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

Parasitism of Meloidogyne eggs by a new fungal parasite

Dactylella oviparasitica, a fungus isolated from Meloidogyne egg masses, was shown to parasitize eggs on agar and in soil, when inoculated as either myceliunt or conidia. The fungus also grew saprophytically on eggs killed ,with methyl bromide or heat. The amount of parasitism in the laboratory suggested that the fungus may be a useful biological control agent against Meloidogyne.     

Zizania latifolia andUstilago esculenta,a Grass-Fungus Association

Zizania latifolia (Manchurian wild rice), native to eastern Asia, is often infected by the smut fungus, Ustilago esculenta, causing culm enlargement and failure to produce flowers. The enlarged infected culms have been used as a vegetable (gau sun) in China since the 10th century. The occasionally-used nameZ. Caduciflora is shown to be only a synonym. To facilitate identification of the host, a taxonomic key shows thatZ. Latifolia differs from and is fully distinct from the American species ofZizania. The nature and structure of the fungus and the history and cultivation of the vegetable are described. As a precaution we propose quarantine of the host and fungus to prevent possible disastrous widespread infection of American wild rices by the fungus.     

Fungal phytopathogen modulates plant and insect responses to promote its dissemination

Vector-borne plant pathogens often change host traits to manipulate vector behavior in a way that favors their spread. By contrast, infection by opportunistic fungi does not depend on vectors, although damage caused by an herbivore may facilitate infection. Manipulation of hosts and vectors, such as insect herbivores, has not been demonstrated in interactions with fungal pathogens. Herein, we establish a new paradigm for the plant-insect-fungus association in sugarcane. It has long been assumed that Fusarium verticillioides is an opportunistic fungus, where it takes advantage of the openings left by Diatraea saccharalis caterpillar attack to infect the plant. In this work, we show that volatile emissions from F. verticillioides attract D. saccharalis caterpillars. Once they become adults, the fungus is transmitted vertically to their offspring, which continues the cycle by inoculating the fungus into healthy plants. Females not carrying the fungus prefer to lay their eggs on fungus-infected plants than mock plants, while females carrying the fungus prefer to lay their eggs on mock plants than fungus-infected plants. Even though the fungus impacts D. saccharalis sex behavior, larval weight and reproduction rate, most individuals complete their development. Our data demonstrate that the fungus manipulates both the host plant and insect herbivore across life cycle to promote its infection and dissemination.Subject terms: Molecular ecology, Molecular ecology     

Arbuscular mycorrhizal fungi reduce the differences in competitiveness between dominant and subordinate plant species

In grassland communities, plants can be classified as dominants or subordinates according to their relative abundances, but the factors controlling such distributions remain unclear. Here, we test whether the presence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices affects the competitiveness of two dominant (Taraxacum officinale and Agrostis capillaris) and two subordinate species (Prunella vulgaris and Achillea millefolium). Plants were grown in pots in the presence or absence of the fungus, in monoculture and in mixtures of both species groups with two and four species. In the absence of G. intraradices, dominants were clearly more competitive than subordinates. In inoculated pots, the fungus acted towards the parasitic end of the mutualism–parasitism continuum and had an overall negative effect on the growth of the plant species. However, the negative effects of the AM fungus were more pronounced on dominant species reducing the differences in competitiveness between dominant and subordinate species. The effects of G. intraradices varied with species composition highlighting the importance of plant community to mediate the effects of AM fungi. Dominant species were negatively affected from the AM fungus in mixtures, while subordinates grew identically with and without the fungus. Therefore, our findings predict that the plant dominance hierarchy may flatten out when dominant species are more reduced than subordinate species in an unfavourable AM fungal relationship (parasitism).     

Ochropsora staphyleae,a new rust pathogen of Japanese bladdernut,found in central Japan

Japanese bladdernut shrub, Staphylea bumalda (Staphyleaceae), was found infected by a fungus in Prefectures of Ibaraki and Tochigi, central Japan. The fungus produced telia beneath the host epidermis and basidiospores on metabasidia emerging from the sori on the abaxial leaf surface. Probasidia were single-celled, sessile, short-cylindrical, thin-walled, and laterally free. A four-celled metabasidium arose from a probasidium by apical elongation. Apparent biotrophic nature and the basidium morphology of the Staphylea fungus suggested its taxonomic affinity to the rust genus Ochropsora. Molecular phylogenetic analyses of D1/D2 region of LSU rDNA showed that two collections of the fungus were grouped with Ochropsora ariae (type species of the genus) and O. nambuana but formed an independent clade. The fungus under consideration was, therefore, classified in Ochropsora, and a new name, O. staphyleae, was proposed for it.     

Acanthocytes of Stropharia rugosoannulata Function as a Nematode-Attacking Device

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.     

Natural infection of the tree hole breeding mosquito Aedes triseriatus (Diptera: Culicidae) with the fungus Funicularius triseriatus

In the course of a study of the population of Aedes triseriatus, a female adults was found to be infected with a fungus. The fungus, Funicularius triseriatus, is described as a new genus and species of the class Hyphomycetes (form-class Deuteromycetes). F. triseriatus is characterized by thallic arthrospores produced on conidiophores arising from hyphae which are funiculose in habit. This is the first report of the natural occurrence of a fungus on A. triseriatus adults.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.