Piriformospora indica-a mutualistic basidiomycete with an exceptionally large plant host range

Piriformospora indica is a basidiomycete of the order Sebacinales, representing a model for the study of mutualistic symbiosis and, beyond that, the plant immune system. The fungus colonizes the roots of a wide range of vascular plants, increasing their growth, seed yield and adaptation to abiotic and biotic stresses. The fungal colonization of roots begins with a biotrophic growth phase, in which living cells are colonized, and continues with a cell death-dependent phase, in which root cells are actively killed by the fungus. The complexity of sebacinalean symbiosis is further enhanced by the presence of endocellular bacteria which may represent significant determinants for a successful outcome of the symbioses. Molecular ecological analyses have revealed an exceptional relevance of sebacinoid fungi in natural ecosystems worldwide. This natural competence could be rooted in their phenotypic adaptability, which, for instance, allows P. indica to grow readily on various synthetic media and to colonize distinct hosts. In molecular and genetic studies, P. indica's mutualistic colonization strategy has been partly unravelled, showing that the jasmonate pathway is exploited for immune suppression and successful development in roots. Research on P. indica supports efforts to make the bioprotective potential of the fungus accessible for agricultural plant production. The decoding of P. indica's genome has revealed its potential for application as bioagent and for targeted improvement of crop plants in biotechnology-based approaches.     

Patterns of interaction between Populus Esch5 and Piriformospora indica: a transition from mutualism to antagonism

Piriformospora indica (Sebacinaceae, Basidiomycota) is an axenically cultivable, plant growth promoting root endophyte with a wide host range, including Populus. Rooting of Populus Esch5 explants started within 6 days after transfer to WPM medium. If such plantlets with roots were inoculated with P. indica, there was an increase in root biomass, and the number of 2nd order roots was increased significantly. A totally different observation was recorded when the explants were placed into WPM with pre-grown P. indica. The interaction led to complete blocking of root production and severely inhibited plant growth. Additionally, branched aerial roots appeared which did not penetrate the medium. On contact with the fungal colony or the medium, the ends of the aerial roots became inflated. Prolonged incubation stimulated the fungus to colonize aerial parts of the plant (stem and leaves). Mycelium not only spread on the surface of the aerial parts, but also invaded the cortical tissues inter- and intracellularly. Detached Populus leaves remained vital for 4 - 5 weeks on sterile agar media or on AspM medium with pre-grown P. indica. When the fungus was pre-grown on culture media such as WPM, containing ammonium as the main source of nitrogen, leaves in contact with the cultures turned brownish within 4 - 12 h. Thereafter, the leaves bleached, and about one day later had become whitish. Thus, cultural conditions could alter the behaviour of the fungus drastically: the outcome of the interaction between plant and fungus can be directed from mutualistic to antagonistic, characterized by fungal toxin formation and extension of the colonization to Populus shoots.     

Specificity in host-fungus associations: Do mutualists differ from antagonists?

Summary Physically intimate interactions between organisms are assumed to be highly specific, yet intimate mutualisms exhibiting little specificity are common and important in many communities. We compare host records for ectomycorrhizal fungi (mutualists) to those for biotrophic shoot fungi and necrotrophic root fungi (both antagonists) in order to test two alternative predictions: (1) intimate physical associations (biotrophy) are more specific than less intimate ones (necrotrophy); (2) antagonisms are more specific than mutualisms. Specificity of fungi for hosts supports prediction (1): ectomycorrhizal fungi and shoot biotrophs are more host specific than root necrotrophs. Fungal symbiont ranges of hosts supports prediction (2): woody hosts are associated with a greater number of mutualistic fungi than antagonistic fungi. The numbers of fungi in the three groups infecting hosts are all significantly positively correlated. This result suggests that some hosts are resistant to fungal invasion and others are quite susceptible. Thus, plants may not be able to erect selective barriers to only antagonistic fungi. The marked asymmetry of specificity from the perspectives of hosts vs fungi suggests that evolutionary and ecological processes act differently on partners in symbioses.     

Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens

Glycosyl hydrolase family 28 (GH28) is a set of structurally related enzymes that hydrolyze glycosidic bonds in pectin, and are important extracellular enzymes for both pathogenic and saprotrophic fungi. Yet, very little is understood about the evolutionary forces driving the diversification of GH28s in fungal genomes. We reconstructed the evolutionary history of family GH28 in fungi by examining the distribution of GH28 copy number across the phylogeny of fungi, and by reconstructing the phylogeny of GH28 genes. We also examined the relationship between lineage-specific GH28 expansions and fungal ecological strategy, testing the hypothesis that GH28 evolution in fungi is driven by ecological strategy (pathogenic vs. non-pathogenic) and pathogenic niche (necrotrophic vs. biotrophic). Our results showed that GH28 phylogeny of Ascomycota and Basidiomycota sequences was structured by specific biochemical function, with endo-polygalacturonases and endo-rhamnogalacturonases forming distinct, apparently ancient clades, while exo-polygalacturonases are more widely distributed. In contrast, Mucoromycotina and Stramenopile sequences formed taxonomically-distinct clades. Large, lineage-specific variation in GH28 copy number indicates that the evolution of this gene family is consistent with the birth-and-death model of gene family evolution, where diversity of GH28 loci within genomes was generated through multiple rounds of gene duplication followed by functional diversification and loss of some gene family members. Although GH28 copy number was correlated with genome size, our findings suggest that ecological strategy also plays an important role in determining the GH28 repertoire of fungi. Both necrotrophic and biotrophic fungi have larger genomes than non-pathogens, yet only necrotrophs possess more GH28 enzymes than non-pathogens. Hence, lineage-specific GH28 expansion is the result of both variation in genome size across fungal species and diversifying selection within the necrotrophic plant pathogen ecological niche. GH28 evolution among necrotrophs has likely been driven by a co-evolutionary arms race with plants, whereas the need to avoid plant immune responses has resulted in purifying selection within biotrophic fungi.     

蓝莓根系复合共生体及其根区土壤中AM真菌调查

蓝莓Vaccinium uliginosum是欧石南菌根（ericoid mycorrhiza,ERM）真菌典型的寄主植物,但同时也可与丛枝菌根（arbuscular mycorrhiza,AM）真菌和深色有隔内生真菌（dark septate endophytes,DSE）共生形成复合共生体。本研究旨在调查和评价不同栽培体制下蓝莓成年树花果期根系共生体发育状况及其根区土壤中AM真菌资源分布状况,以期为优质蓝莓栽培管理提供理论依据和技术基础。从青岛佳沃蓝莓基地采集暖棚、冷棚和露地3种方式栽培的9-10年生‘蓝丰’、‘奥尼尔’和‘公爵’蓝莓的根系及根区土样,观察测定根系共生体着生数量、根区土壤中AM真菌孢子数量和菌种组成。结果表明,所有栽培方式下供试品种蓝莓根系均形成ERM、AM和DSE结构及其复合共生体;其中,AM着生数量最多,其次是ERM,DSE侵染率最低;复合共生体中则呈现ERM+AM>ERM+DSE>ERM+AM+DSE;蓝莓复合共生体着生数量、AM真菌侵染率、丛枝着生率及孢子数量等不同种植方式下呈现暖棚>冷棚>露地,不同品种呈现‘蓝丰’>‘公爵’>‘奥尼尔’,而ERM和DSE侵染率也呈现上述变化趋势。依据AM真菌形态特征,供分离鉴定获得5属11种AM真菌,以暖棚栽培条件下分离获得的AM真菌数量最多,‘蓝丰’根区土壤中分布的AM真菌属种最多。暖棚内成年树花果期蓝莓根系共生体发育健全,AM真菌种类和孢子数量较多,可能有利于提高蓝莓的产量、改善果实品质和抗逆性。     

Landscape genomics of an obligate mutualism: Concordant and discordant population structures between the leafcutter ant Atta texana and its two main fungal symbiont types

To explore landscape genomics at the range limit of an obligate mutualism, we use genotyping‐by‐sequencing (ddRADseq) to quantify population structure and the effect of host–symbiont interactions between the northernmost fungus‐farming leafcutter ant Atta texana and its two main types of cultivated fungus. Genome‐wide differentiation between ants associated with either of the two fungal types is of the same order of magnitude as differentiation associated with temperature and precipitation across the ant's entire range, suggesting that specific ant–fungus genome–genome combinations may have been favoured by selection. For the ant hosts, we found a broad cline of genetic structure across the range, and a reduction of genetic diversity along the axis of range expansion towards the range margin. This population‐genetic structure was concordant between the ants and one cultivar type (M‐fungi, concordant clines) but discordant for the other cultivar type (T‐fungi). Discordance in population‐genetic structures between ant hosts and a fungal symbiont is surprising because the ant farmers codisperse with their vertically transmitted fungal symbionts. Discordance implies that (a) the fungi disperse also through between‐nest horizontal transfer or other unknown mechanisms, and (b) genetic drift and gene flow can differ in magnitude between each partner and between different ant–fungus combinations. Together, these findings imply that variation in the strength of drift and gene flow experienced by each mutualistic partner affects adaptation to environmental stress at the range margin, and genome–genome interactions between host and symbiont influence adaptive genetic differentiation of the host during range evolution in this obligate mutualism.     

Secondary Metabolism and Biotrophic Lifestyle in the Tomato Pathogen Cladosporium fulvum

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.     

Isolation and Characterisation of Pitef1 Encoding the Translation Elongation Factor EF-1α of the Root Endophyte Piriformospora indica

Abstract: Piriformospora indica (Hymenomycetes, Basidiomycota) is a newly described endophyte which interacts with the roots of a great variety of plants, showing a positive effect on biomass production. In order to obtain a tool for molecular studies on P. indica, Pitef 1 encoding the translation elongation factor EF-1α in P. indica has been cloned and analysed. Comparison of the genomic and cDNA sequence revealed the presence of seven introns in the coding part of the gene and at least one in the 5'untranslated region. Pitef 1 is only present as one copy in the genome, as determined by Southern blot analysis. Interaction with roots of Zea mays in a time course experiment was analysed in relation to hyphal development and RNA accumulation, showing high expression of this gene. The Pitef 1 promoter should therefore be a good tool to construct vectors for the development of a transformation system for P. indica. The gene Pitef 1 might, in addition, be useful for estimating the amount of active mycelium during in planta development and for the calibration of RNA accumulation analyses of differentially expressed fungal genes.     

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.     

Piriformospora indica: a versatile root endophytic symbiont

Piriformospora indica, a unique root endophytic basidiomycete, exhibits symbiotic association with a variety of host plants. Spacio-temporally, the biphasic lifestyle of the fungus is characterized by distinct biotrophic and cell death associated root colonization processes. Fungal components such as fungal chitin induce specific host immune responses which also play an important role in establishing a successful sustainable interaction. The fungus-induced Pattern Triggered Immunity (PTI) is regulated by a complex interplay amongst the various phytohormones and anti-microbial molecules. The association so built up imparts a broad spectrum of benefits to the host viz., high salt tolerance, disease resistance, resistance to heavy metal toxicity, and growth-promotion involving value addition. These characteristics of P. indica can be translated into specific applications in agriculture and horticulture. The current article also outlines the future research prospects of this highly versatile fungus.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

Challenges and progress towards understanding the role of effectors in plant-fungal interactions

Both mutualistic and biotrophic pathogenic fungi rely on living host plants for growth and reproduction and must modify host cell structure and function for successful infection. The deployment of a diverse set of secreted virulence determinants referred to as 'effectors', many of which are directly delivered into the host cell, is postulated to be the key to host infection. This review provides a snapshot of the current progress in fungal effector biology. Recent genome sequencing of rust and powdery mildew obligate biotrophs has provided insight into the repertoires of potential effectors of these highly specialised pathogens. Identification of the first host-translocated effectors from mutualistic fungi has revealed that these fungi also manipulate host cells through effectors. The biological activities of some fungal effectors are just beginning to be revealed, while much uncertainty still surrounds the mechanisms of transport into host cells.     

Genomes, free radicals and plant cell invasion: recent developments in plant pathogenic fungi

This review describes current advances in our understanding of fungal-plant interactions. The widespread application of whole genome sequencing to a diverse range of fungal species has allowed new insight into the evolution of fungal pathogenesis and the definition of the gene inventories associated with important plant pathogens. This has also led to functional genomic approaches to carry out large-scale gene functional analysis. There has also been significant progress in understanding appressorium-mediated plant infection by fungi and its underlying genetic basis. The nature of biotrophic proliferation of fungal pathogens in host tissue has recently revealed new potential mechanisms for cell-to-cell movement by invading pathogens.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.