Phosphate import at the arbuscule: just a nutrient?

Central to the mutualistic arbuscular mycorrhizal symbiosis is the arbuscule, the site where symbiotic phosphate is delivered. Initial investigations in legumes have led to the exciting observation that symbiotic phosphate uptake not only enhances plant growth but also regulates arbuscule dynamics and is, furthermore, required for maintenance of the symbiosis. This review evaluates the possible role of the phosphate ion, not only as a nutrient but also as a signal that is necessary for reprogramming the host cortex cell for symbiosis.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization.

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family.

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.

Nonself vegetative fusion and genetic exchange in the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants and form extensive underground hyphal networks simultaneously connecting the roots of different plant species. No empirical evidence exists for either anastomosis between genetically different AMF or genetic exchange.Five isolates of one population of Glomus intraradices were used to study anastomosis between hyphae of germinating spores. We show that genetically distinct AMF, from the same field, anastomose, resulting in viable cytoplasmic connections through which genetic exchange could potentially occur.Pairs of genetically different isolates were then co-cultured in an in vitro system.Freshly produced spores were individually germinated to establish new cultures.Using several molecular tools, we show that genetic exchange occurred between genetically different AMF. Specific genetic markers from each parent were transmitted to the progeny. The progeny were viable, forming symbioses with plant roots. The phenotypes of some of the progeny were significantly different from either parent.Our results indicate that considerable promiscuity could occur in these fungi because nine out of 10 combinations of different isolates anastomosed. The ability to perform genetic crosses between AMF experimentally lays a foundation for understanding the genetics and evolutionary biology of these important plants symbionts.

Different arbuscular mycorrhizal fungi alter coexistence and resource distribution between co-occurring plants

It is often thought that the coexistence of plants and plant diversity is determined by resource heterogeneity of the abiotic environment. However, the presence and heterogeneity of biotic plant resources, such as arbuscular mycorrhizal fungi (AMF), could also affect plant species coexistence. In this study, Brachypodium pinnatum and Prunella vulgaris were grown together in pots and biotic resource heterogeneity was simulated by inoculating these pots with one of three different AMF taxa, with a mixture of these three taxa, or pots remained uninoculated. The AMF acted as biotic plant resources since the biomass of plants in pots inoculated with AMF was on average 11.8 times higher than uninoculated pots. The way in which the two plant species coexisted, and the distribution of phosphorus and nitrogen between the plant species, varied strongly depending on which AMF were present. The results showed that the composition of AMF communities determines how plant species coexist and to which plant species nutrients are allocated. Biotic plant resources such as AMF should therefore be considered as one of the factors that determine how plant species coexist and how soil resources are distributed among co-occurring plant species.

Arbuscular mycorrhizal fungi (Glomeromycota) harbour ancient fungal tubulin genes that resemble those of the chytrids (Chytridiomycota).

The genes encoding alpha- and beta-tubulins have been widely sampled in most major fungal phyla and they are useful tools for fungal phylogeny. Here, we report the first isolation of alpha-tubulin sequences from arbuscular mycorrhizal fungi (AMF). In parallel, AMF beta-tubulins were sampled and analysed to identify the presence of paralogs of this gene. The AMF alpha-tubulin amino acid phylogeny was congruent with the results previously reported for AMF beta-tubulins and showed that AMF tubulins group together at a basal position in the fungal clade and showed high sequence similarities with members of the Chytridiomycota. This is in contrast with phylogenies for other regions of the AMF genome. The amount and nature of substitutions are consistent with an ancient divergence of both orthologs and paralogs of AMF tubulins. At the amino acid level, however, AMF tubulins have hardly evolved from those of the chytrids. This is remarkable given that these two groups are ancient and the monophyletic Glomeromycota probably diverged from basal fungal ancestors at least 500 million years ago. The specific primers we designed for the AMF tubulins, together with the high molecular variation we found among the AMF species we analysed, make AMF tubulin sequences potentially useful for AMF identification purposes.

Genetic variability in Arbuscular Mycorrhizal Fungi : effect on gene transcription of "Oryza Sativa"

AbstractArbuscular Mycorrhizal Fungi (AMF) form obligate symbioses with the majority of land plants. These fungi influence the diversity and productivity of plants. AMF are unusual organisms, harbouring genetically different nuclei in a common cytoplasm (known as heterokaryosis). Genetic variability has been shown between AMF individuals coming from the same population. Recent findings showed that genetic exchange between genetically different AMF individuals was possible. Additionnaly, segregation was shown to occur at spore formation in AMF. These two processes were shown to increase genetic variability between AMF individuals.Because of the difficulty to study these organisms, almost nothing is known about the effect of intra-specific genetic variability in AMF on the plant transcriptome. The aim of this thesis was to bring insights into the effect of intra-specific genetic variability in AMF on plant gene transcription. We demonstrated that genetic exchange could influence expression of some symbiosis specific plant genes and the timing of the colonization of the fungi in plant roots. We also showed that segregation could have a large impact on plant gene transcription. Taken together, these results demonstrated that AMF intra-specific variability could profoundly affect the life of plants by altering various molecular pathways. Moreover, results obtained on rice open a field of research on AMF genetics in impromvment of growth in agricultural plants and should be taken into account for future experiments.RésuméLes champignons endomycorhiziens arbusculaires (CEA) forment une symbiose obligatoire avec la majorité des plantes sur terre. Ces champignons peuvent influencer la diversité et la productivité des plantes avec lesquelles ils forment la symbiose. Les CEA sont des organismes particuliers de part le fait qu'ils possèdent des noyaux génétiquement différents (appelés hétérocaryosis) dans un cytoplasme commun. Il a été montré qu'il existait de la variabilité génétique intra-specific chez les CEA. De plus, des études recentes ont montré que l'échange génétique chez les CEA était possible entre des individus génétiquement différents tout comme la ségrégation qui a aussi été démontrée au moment de la formation des nouvelles spores chez les CEA. Ces deux processus ont été montrés comme pouvant créer aussi de la variabilité génétique intra-specific.Du fait de la difficulté de travailler avec les CEA et à cause de la nouveauté de ces recherches, très peu de choses sont connues sur l'effet de l'échange génétique et de la ségrégation chez les CEA sur les plantes, et particulièrement au niveau moléculaire. Le but de cette thèse a été d'apporter la lumière sur les effets de la viariabilité génétique intra-specific chez les CEA, sur la transcription des gènes chez la plante. Nous avons pu montrer que l'échange génétique pouvait avoir des effets sur l'expression de gènes spécifiques à cette symbiose mais aussi pouvait influencer le timing de colonisation des racines de plantes par les CEA. Nous avons aussi montré que la ségrégation pouvait grandement influencer le transcriptome complet de la plante, et pas seulement les voies métaboliques spécifiques à la symbiose comme cela avait été montré auparavant.L'ensemble de ces résultats démontre l'importance de la variation intra-specific chez les CEA sur les plantes et leur implication sur leur cycle de vie en changeant l'expression de voies métaboliques. De plus, ces résultats obtenus sur le riz ouvrent un champ de recherches sur les plantes destinées à l'agriculture et devraient être pris en compte pour des expériences futures.

Relatedness among arbuscular mycorrhizal fungi drives plant growth and intraspecific fungal coexistence.

Arbuscular mycorrhizal fungi (AMF) form symbioses with most plant species. They are ecologically important determinants of plant growth and diversity. Considerable genetic variation occurs in AMF populations. Thus, plants are exposed to AMF of varying relatedness to each other. Very little is known about either the effects of coexisting AMF on plant growth or which factors influence intraspecific AMF coexistence within roots. No studies have addressed whether the genetics of coexisting AMF, and more specifically their relatedness, influences plant growth and AMF coexistence. Relatedness is expected to influence coexistence between individuals, and it has been suggested that decreasing ability of symbionts to coexist can have negative effects on the growth of the host. We tested the effect of a gradient of AMF genetic relatedness on the growth of two plant species. Increasing relatedness between AMFs lead to markedly greater plant growth (27% biomass increase with closely related compared to distantly related AMF). In one plant species, closely related AMF coexisted in fairly equal proportions but decreasing relatedness lead to a very strong disequilibrium between AMF in roots, indicating much stronger competition. Given the strength of the effects with such a shallow relatedness gradient and the fact that in the field plants are exposed to a steeper gradient, we consider that AMF relatedness can have a strong role in plant growth and the ability of AMF to coexist. We conclude that AMF relatedness is a driver of plant growth and that relatedness is also a strong driver of intraspecific coexistence of these ecologically important symbionts.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

? The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. ? We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. ? We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. ? Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Plant and arbuscular mycorrhizal fungal diversity - are we looking at the relevant levels of diversity and are we using the right techniques?

Fungal symbionts commonly occur in plants influencing host growth, physiology, and ecology (Carlile et al., 2001). However, while whole-plant growth responses to biotrophic fungi are readily demonstrated, it has been much more difficult to identify and detect the physiological mechanisms responsible. Previous work on the clonal grass Glyceria striata has revealed that the systemic fungal endophyte Epichloë glyceriae has a positive effect on clonal growth of its host (Pan & Clay, 2002; 2003). The latest study from these authors, in this issue (pp. 467- 475), now suggests that increased carbon movement in hosts infected by E. glyceriae may function as one mechanism by which endophytic fungi could increase plant growth. Given the widespread distribution of both clonal plants and symbiotic fungi, this research will have implications for our understanding of the ecology and evolution of fungus-plant associations in natural communities.

A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants.

BACKGROUND: Genes involved in arbuscular mycorrhizal (AM) symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes (forward genetics). In addition, a number of AM-related genes has been identified by their AM-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches (reverse genetics). However, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. If such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. The goal of this study was to systematically search for AM-related genes with a bioinformatics strategy that is insensitive to these problems. The central element of our approach is based on the fact that many AM-related genes are conserved only among AM-competent species. RESULTS: Our approach involves genome-wide comparisons at the proteome level of AM-competent host species with non-mycorrhizal species. Using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the AM-competent species. Proteins of these clusters were then analyzed in an extended set of 16 plant species and ranked based on their relatedness among AM-competent monocot and dicot species, relative to non-mycorrhizal species. In addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. As a result we present a list of yet uncharacterized proteins that show a strongly AM-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in AM. Among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the S-locus receptor kinases involved in sporophytic self-incompatibility. CONCLUSIONS: We present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. This strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest.

Evolutionary genetics of arbuscular mycorrhizal fungi - causes and consequences of multiple genomes in Glomus intraradices

Résumé Les champignons endomycorhiziens arbusculaires (CEA) forment des symbioses avec la plupart des plantes terrestres. Les CEA influencent la croissance des plantes et la biodiversité. Ils sont supposés avoir évolué de manière asexuée pendant au moins 400 millions d'années et aucune diversification morphologique majeure n'a été constatée. Pour ces raisons, les CEA sont considérés comme d'anciens asexués. Très peu d'espèces sont connues actuellement. Les individus de ces champignons contiennent des noyaux génétiquement différents dans un cytoplasme continu. La signification évolutive, la variabilité et la maintenance des génomes multiples au sein des individus sont inconnues. Ce travail a démontré qu'une population du CEA Glomus intraradices est génétiquement très variable. Nous avons conclu que les plantes hôtes plutôt que la différenciation géographique devraient être responsables de cette grande diversité. Puis nous avons cherché l'existence de recombinaison entre génotypes dans une population. Nous avons détecté un groupe recombinant au sein de la population, ce qui met en doute l'état d'anciens asexués des CEA. Nous avons également détecté l'occurrence de fusions d'hyphes et l'échange de noyaux entre isolats génétiquement différents. La descendance hybride issue de cet échange était viable et distincte phénotypiquement des isolats parentaux. En résumé, ce travail identifie des événements cruciaux dans le cycle de vie des CEA qui ont le potentiel d'influencer l'évolution de génomes multiples. L'étude des conséquences de ces événements sur les interactions avec les plantes hôtes pourrait éclaircir significativement la compréhension de la symbiose entre plantes et CEA. Abstract Arbuscular mycorrhizal fungi (AMF) are important symbionts of most land plants. AMF influence plant growth and biodiversity. Very few extant species are described. AMF are thought to have evolved asexually for at least 400 million years and no major morphological diversification has occurred. Due to these reasons, they were termed `ancient asexuals'. Fungal individuals harbour genetically different nuclei in a continuous cytoplasm. The variability, maintenance and evolutionary significance of multiple genomes within individuals are unknown. This work showed that a population of the AMF Glomus intraradices harbours very high genetic diversity. We concluded that host plants rather than geographic differentiation were responsible for this diversity. Furthermore, we investigated whether recombination occurred among genotypes of a G. intraradices population. The identification of a core group of recombining genotypes in the population refutes the assumption of ancient asexuality in AMF. We found that genetically different isolates can form hyphal fusions and exchange nuclei. The hybrid progeny produced by the exchange was viable and phenotypically distinct from the parental isolates. Taken together, this work provided evidence for key events in the AMF life cycle, that influence the evolution of multiple genomes. Studying the consequences of these events on the interaction with host plants may significantly further the understanding of the AMF-plant symbiosis.

Weights in the balance: jasmonic acid and salicylic acid signaling in root-biotroph interactions.

Work on the interaction of aerial plant parts with pathogens has identified the signaling molecules jasmonic acid (JA) and salicylic acid (SA) as important players in induced defense of the plant against invading organisms. Much less is known about the role of JA and SA signaling in root infection. Recent progress has been made in research on plant interactions with biotrophic mutualists and parasites that exclusively associate with roots, namely arbuscular mycorrhizal and rhizobial symbioses on one hand and nematode and parasitic plant interactions on the other hand. Here, we review these recent advances relating JA and SA signaling to specific stages of root colonization and discuss how both signaling molecules contribute to a balance between compatibility and defense in mutualistic as well as parasitic biotroph-root interactions.

Evidence for the evolution of multiple genomes in arbuscular mycorrhizal fungi.

Ancient asexuals directly contradict the evolutionary theories that explain why organisms should evolve a sexual life history. The mutualistic, arbuscular mycorrhizal fungi are thought to have been asexual for approximately 400 million years. In the absence of sex, highly divergent descendants of formerly allelic nucleotide sequences are thought to evolve in a genome. In mycorrhizal fungi, where individual offspring receive hundreds of nuclei from the parent, it has been hypothesized that a population of genetically different nuclei should evolve within one individual. Here we use DNA-DNA fluorescent in situ hybridization to show that genetically different nuclei co-exist in individual arbuscular mycorrhizal fungi. We also show that the population genetics techniques used in other organisms are unsuitable for detecting recombination because the assumptions and underlying processes do not fit the fungal genomic structure shown here. Instead we used a phylogenetic approach to show that the within-individual genetic variation that occurs in arbuscular mycorrhizal fungi probably evolved through accumulation of mutations in an essentially clonal genome, with some infrequent recombination events. We conclude that mycorrhizal fungi have evolved to be multi-genomic.

Ecology and evolution of multigenomic arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) form extremely important mutualistic symbioses with most plants. Their role in nutrient acquisition, plant community structure, plant diversity, and ecosystem productivity and function has been demonstrated in recent years. New findings on the genetics and biology of AMF also give us a new picture of how these fungi exist in ecosystems. In this article, I bring together some recent findings that indicate that AMF have evolved to contain multiple genomes, that they connect plants together by a hyphal network, and that these different genomes may potentially move around in this network. These findings show the need for more intensive studies on AMF population biology and genetics in order to understand how they have evolved with plants, to better understand their ecological role, and for applying AMF in environmental management programs and in agriculture. A number of key features of AMF population biology have been identified for future studies and most of these concern the need to understand drift, selection, and genetic exchange in multigenomic organisms, a task that has not previously presented itself to evolutionary biologists.