Plant-microbe Symbiosis Research Articles

Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution.

Mutualistic symbioses between eukaryotes and beneficial microorganisms of their microbiome play an essential role in nutrition, protection against disease, and development of the host. However, the impact of beneficial symbionts on the evolution of host genomes remains poorly characterized. Here we used the independent loss of the most widespread plant–microbe symbiosis, arbuscular mycorrhization (AM), as a model to address this question. Using a large phenotypic approach and phylogenetic analyses, we present evidence that loss of AM symbiosis correlates with the loss of many symbiotic genes in the Arabidopsis lineage (Brassicales). Then, by analyzing the genome and/or transcriptomes of nine other phylogenetically divergent non-host plants, we show that this correlation occurred in a convergent manner in four additional plant lineages, demonstrating the existence of an evolutionary pattern specific to symbiotic genes. Finally, we use a global comparative phylogenomic approach to track this evolutionary pattern among land plants. Based on this approach, we identify a set of 174 highly conserved genes and demonstrate enrichment in symbiosis-related genes. Our findings are consistent with the hypothesis that beneficial symbionts maintain purifying selection on host gene networks during the evolution of entire lineages.

Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes

Perennial ryegrass (Lolium perenne) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3–25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.

Does Fungal Endophyte Infection Improve Tall Fescue’s Growth Response to Fire and Water Limitation?

Invasive species may owe some of their success in competing and co-existing with native species to microbial symbioses they are capable of forming. Tall fescue is a cool-season, non-native, invasive grass capable of co-existing with native warm-season grasses in North American grasslands that frequently experience fire, drought, and cold winters, conditions to which the native species should be better-adapted than tall fescue. We hypothesized that tall fescue’s ability to form a symbiosis with Neotyphodium coenophialum, an aboveground fungal endophyte, may enhance its environmental stress tolerance and persistence in these environments. We used a greenhouse experiment to examine the effects of endophyte infection (E+ vs. E−), prescribed fire (1 burn vs. 2 burn vs. unburned control), and watering regime (dry vs. wet) on tall fescue growth. We assessed treatment effects for growth rates and the following response variables: total tiller length, number of tillers recruited during the experiment, number of reproductive tillers, tiller biomass, root biomass, and total biomass. Water regime significantly affected all response variables, with less growth and lower growth rates observed under the dry water regime compared to the wet. The burn treatments significantly affected total tiller length, number of reproductive tillers, total tiller biomass, and total biomass, but treatment differences were not consistent across parameters. Overall, fire seemed to enhance growth. Endophyte status significantly affected total tiller length and tiller biomass, but the effect was opposite what we predicted (E−>E+). The results from our experiment indicated that tall fescue was relatively tolerant of fire, even when combined with dry conditions, and that the fungal endophyte symbiosis was not important in governing this ecological ability. The persistence of tall fescue in native grassland ecosystems may be linked to other endophyte-conferred abilities not measured here (e.g., herbivory release) or may not be related to this plant-microbial symbiosis.

Inside Arbuscular Mycorrhizal Roots – Molecular Probes to Understand the Symbiosis

Associations between arbuscular mycorrhizal (AM) fungi and plants are an ancient and widespread plant microbe symbioses. Most land plants can associate with this specialized group of soil fungi (in the Glomeromycota), which enhance plant nutrient uptake in return for C derived from plant photosynthesis. Elucidating the mechanisms involved in the symbiosis between obligate symbionts such as AM fungi and plant roots is challenging because AM fungal transcripts in roots are in low abundance and reference genomes for the fungi have not been available. A deep sequencing metatranscriptomics approach was applied to a wild‐type tomato and a tomato mutant (Solanum lycopersicum L. cultivar RioGrande 76R) incapable of supporting a functional AM symbiosis, revealing novel AM fungal and microbial transcripts expressed in colonized roots. We confirm transcripts known to be mycorrhiza associated and report the discovery of more than 500 AM fungal and novel plant transcripts associated with mycorrhizal tomato roots including putative Zn, Fe, aquaporin, and carbohydrate transporters as well as mycorrhizal‐associated alternative gene splicing. This analysis provides a fundamental step toward identifying the molecular mechanisms of mineral and carbohydrate exchange during the symbiosis. The utility of this metatranscriptomic approach to explore an obligate biotrophic interaction is illustrated, especially as it relates to agriculturally relevant biological processes.

Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25–60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg·kg−1 Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mg·kg−1 Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2–80.5%) in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation.

The molecular basis for construction of highly productive ecologically sustainable agrocenoses

The methodology is suggested for analyzing the variability and heredity in super-organism genetic systems of different complexity ranging from two-component plant-microbe and animal-microbe symbiosis to the endosymbiotic and soil microbe communities. The approaches based on symbiogenetics and metagenomics may be used for development of highly productive ecologically sustainable agrocenoses based on the substitution of agrochemicals (mineral fertilizers, pesticides) by microbe preparations. A possibility to use the natural analogs of agrocenoses for creation of the models that allowed performing the directed construction and improvement of productivity in the sustainable agricultural systems is emphasized.

Coevolution of partners and integrity of symbiotic systems

Symbioses are very beneficial models for studying the integrity of biosystems, which characterizes their structure-function orderliness and allows the partners to adequately respond to environmental changes. An analysis of various types of the plant-microbe and animal-microbe symbioses suggests that their integrity qualitatively increases during facultative and ecologically obligatory interactions of the partners, reaching its climax in the case of genetically obligatory interactions. Mathematical models enabled us to demonstrate that the functional integrity of N2-fixing legume-rhizobium symbiosis (concordance of the changes in the genotypic frequencies of the partners induced by environmental fluctuations) correlates with its ecological efficiency, which is increased by natural selection. The result is the establishment of tight regulatory feedbacks between the partners, leading to their genetic integration, which is referred to as “symbiogenome.” The genetic integrity of symbiosis determines its high evolutionary potential based on (a) the epigenetic inheritance of symbiotic characters by the host, implemented as a vertical transfer of either microsymbionts themselves or their genes and (b) interspecific altruism, which is associated with positive feedbacks between the partners and determines the ecological efficiency of mutualistic interactions. Realization of this potential leads to the deep genetic integration of initially independent partners, including their fusion into novel integral organisms.

Biochar enhances seedling growth and alters root symbioses and properties of sub-boreal forest soils

Robertson, S. J., Rutherford, P. M., López-Gutiérrez, J. C. and Massicotte, H. B. 2012. Biochar enhances seedling growth and alters root symbioses and properties of sub-boreal forest soils. Can. J. Soil Sci. 92: 329–340. Biochar application may enhance soil properties, improve plant productivity, and increase long-term carbon storage, but impacts of biochar on plant-microbe symbioses mediating plant nutrient uptake in temperate or boreal soils are not well known. We planted lodgepole pine (Pinus contorta var. latifolia) or sitka alder (Alnus viridis ssp. sinuata) seeds in pots containing field-collected forest soils (from central British Columbia) amended with 0, 5, or 10% (dry mass basis) biochar with and without urea fertilizer (150 mg N kg−1). Pine seedlings were harvested at 4 mo and roots were assessed for abundance and diversity of ectomycorrhizal (ECM) morphotypes using light microscopy and DNA sequencing. Biochar raised soil pH, exchangeable cations and cation exchange capacity in some treatments in both soils. Pine had greater biomass in biochar+fertilizer treatments compared to control and fertilizer-only treatments; this corresponded in part to an increase in abundance of some ECMs. Alder seedlings were harvested at 2, 3 and 4 mo to measure N fixation in root nodules using acetylene reductase assay (ARA). Alder seedlings had greater shoot biomass when grown in biochar-amended soils compared with unamended control. Although mean ARA rates (at 4 mo) were greater in biochar-amended soils compared with controls, the data showed great variation and differences were not statistically significant (P>0.05). This study showed that biochar addition can enhance soil properties and the early growth of pine and alder in some sub-boreal forest soils; small changes in ECM abundances may be expected.

Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture

Plant growth-promoting rhizobacteria (PGPR) are the rhizosphere bacteria that can enhance plant growth by a wide variety of mechanisms like phosphate solubilization, siderophore production, biological nitrogen fixation, rhizosphere engineering, production of 1-Aminocyclopropane-1-carboxylate deaminase (ACC), quorum sensing (QS) signal interference and inhibition of biofilm formation, phytohormone production, exhibiting antifungal activity, production of volatile organic compounds (VOCs), induction of systemic resistance, promoting beneficial plant-microbe symbioses, interference with pathogen toxin production etc. The potentiality of PGPR in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements. Growth promoting substances are likely to be produced in large quantities by these rhizosphere microorganisms that influence indirectly on the overall morphology of the plants. Recent progress in our understanding on the diversity of PGPR in the rhizosphere along with their colonization ability and mechanism of action should facilitate their application as a reliable component in the management of sustainable agricultural system. The progress to date in using the rhizosphere bacteria in a variety of applications related to agricultural improvement along with their mechanism of action with special reference to plant growth-promoting traits are summarized and discussed in this review.

Focus on Symbioses: “Where Two Are Better Than One”

In the last two decades, tremendous advances have been made in our understanding of the basic principles governing two agriculturally relevant symbioses of plants with beneficial microorganisms: i) the nitrogen-fixing nodule bacteria of legumes and angiosperms and ii) phosphate-acquiring mycorrhiza fungi. This special focus issue brings together articles covering aspects of evolution and phylogeny, diversity, signaling, differentiation, and development of new investigative tools. Together they provide a window into the exciting world of plant-microbe symbioses.

Legume root symbioses: Natural history and prospects for improvement

Legumes develop different mutually beneficial microbial-root symbioses such as arbuscular mysorrhiza (AM), rhizobium-legume symbiosis (RLS) and epiphytic or endophytic associations with plant growth-promoting bacteria (PGPB) which are distinguished in level of integration of the partners. Evidences of the role of AM as ancestral form of symbiosis which might be a source of the legume pre-adaptation to form some RLS are demonstrated. The RLS is supposed to evolve for a few times in ancient legumes in parallel ways based on the universal organization and regulatory mechanisms of the plant genetic material. Associations of plant roots with PGPB probably are the vestige of the early stages of evolution in morphologically differentiated RLS. Also, it is quite possible that 'first' rhizobia have originated from bacterial endosymbionts of AM fungi; then AM fungi might operate as effective vectors for introducing bacteria into the plants. Thus, the legume root symbioses may be considered as a single 'evolutionary plant-microbial continuum'. The acquired knowledge about evolution of plantmicrobe symbioses would contribute to the creation of new commercial varieties of plants with the use of both bio-engineered methods and traditional plant breeding. An original conception of legume breeding to improve their symbiotic effectiveness is proposed.

Understanding context-dependency in plant–microbe symbiosis: The influence of abiotic and biotic contexts on host fitness and the rate of symbiont transmission

Understanding the dynamics of a hereditary symbiosis requires testing how ecological factors alter not only the fitness consequences of the symbiosis, but also the rate of symbiont transmission to the next generation. The relative importance of these two mechanisms remains unresolved because studies have not simultaneously examined how the ecological context of the symbiosis influences both costs/benefits and the rate of vertical transmission. Fungal endophytes in grasses have provided particularly tractable systems for investigating the ecological and evolutionary dynamics of hereditary symbiosis. Here we examine interactions between a fungal endophyte, Epichloë amarillans, and its grass host, Agrostis hyemalis, under altered abiotic and biotic contexts: a gradient of water availability and in the presence versus absence of soil microbes. We show that benefits of the symbiosis were strongest when water was limiting. Symbiotic plants at the lowest watering level produced ∼40% more inflorescences and greater seed mass than non-symbiotic plants, while at the highest watering level, symbiotic and non-symbiotic plants did not significantly differ in reproductive fitness. Benefits appear to accrue by allowing hosts to escape from drought, a response that has not been previously reported to be endophyte-mediated. Symbiotic plants at the lowest watering level flowered 9 days earlier than non-symbiotic plants. Interestingly, our results suggest the symbiosis may be costly in the presence of soil microbes, as on live soil, the biomass of symbiotic plants was lower than the biomass of symbiont-free plants. We detected no effect of either the biotic or abiotic context on the rate of symbiont vertical transmission, suggesting that the context-dependent benefits of the symbiosis are the more important driver of variation in symbiont frequency in this system.

Deciphering the relative contributions of multiple functions within plant–microbe symbioses

For microbial symbioses with plants, such as mycorrhizas, we typically quantify either the net effects of one partner on another or a single function a symbiont provides. However, many microbial symbioses provide multiple functions to plants that vary based on the microbial species or functional group, plant species, and environment. Here we quantified the relative contributions of multiple functions provided by arbuscular mycorrhizal (AM) fungi to symbiont-mediated changes in plant biomass. We used two published data sets, one that measured multiple functions (pathogen protection and nutrient uptake) on a single plant and one that measured a single function (pathogen protection) on multiple plants. Using structural equation modeling, we observed strong variation in the functional pathways by which AM fungi altered plant growth; changes in plant biomass were associated with different functions (and different AM fungal functional groups) for the different plant species. Utilizing this methodology across multiple partners and environments will allow researchers to gauge the relative importance of functions they isolate and, perhaps more importantly, those they did not consider. This baseline information is essential for establishing the specific mechanisms by which microbial symbioses influence plant diversity and to more effectively utilize these organisms in agriculture, restoration and conservation.

Deciphering the Relative Contributions of Multiple Functions within Plant-Microbe Symbioses

Deciphering the Relative Contributions of Multiple Functions within Plant-Microbe Symbioses

Large-Scale Phosphoprotein Analysis inMedicago truncatulaRoots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.

Epigenetic modification of rhizobial genome is essential for efficient nodulation

CcrM is one of the solitary bacterial DNA methyltransferases which does not have corresponding restriction enzymes. We established a stable ccrM-overexpressing mutant of Mesorhizobium loti, MlccrM-OX, and performed molecular and phenotypic characterization of this strain. In the M. loti MlccrM-OX infected plants, nodulation was apparently delayed at 7 days after inoculation (dai), however, the nodules that eventually formed on the MlccrM-OX roots showed nitrogen fixing ability by at least 21 dai. These results suggest that the initial morphogenic events were affected by ccrM-overexpression and that the correct pattern of DNA methylation of the bacterial genome is not essential for plant–microbe symbiosis, but are required for efficient nodulation.

Host plant as an organizer of microbial evolution in the beneficial symbioses

Evolution of beneficial plant–microbe symbioses is presented as a result of selective processes induced by hosts in the associated microbial populations. These processes ensure a success of “genuine mutualists” (which benefit the host, often at the expense of their own fitness) in competition with “symbiotic cheaters” (which consume the resources provided by host without expressing the beneficial traits). Using a mathematical model describing the cyclic microevolution of rhizobia–legume symbiosis, we suggest that the selective pressures in favor of N2-fixing (Fix+) strains operate within the in planta bacterial population due to preferential allocation of C resources into Fix+ nodules (positive partners’ feedbacks). Under the clonal infection of nodules, Fix+ strains (“genuine mutualists”) are supported by the group (inter-deme, kin) selection while under the mixed infections, this selection is ineffective since the Fix+ strains are over-competed by Fix− ones (“symbiotic cheaters”) in the nodular habitats. Nevertheless, under mixed infections, Fix+ strains may be supported due to the coevolutionary responses form plant population which induce the mutualism-specific types of natural (group, individual) selection including the frequency dependent selection implemented in rhizobia population during the competition for host infection. Using the model of multi-strain bacterial competition for inoculation of symbiotic (rhizospheric, nodular) habitats, we demonstrate that the individual selection in favor of host-specific mutualist genotypes is more intensive than in favor of non-host-specific genotypes correlating the experimental data on the coordinated increases of symbiotic efficiency and specificity in the rhizobia–legume coevolution. However, an overall efficiency of symbiotic system is maximal when the non-host-specific mutualists are present in rhizobia population, and selection in favor of these mutualists operating at the whole population level is of key importance for improving the symbiosis. Construction of the agronomically valuable plant–microbe systems should provide the optimization of host-specific versus non-host-specific mutualists’ composition in legume inoculants combined with the clonal penetration of these mutualists into the nodules.

From plant–microbe interactions to symbiogenetics: a universal paradigm for the interspecies genetic integration

AbstractBeneficial plant–microbe symbioses are based on the integration of genetic material from diverse organisms resulting in formation of superorganism genetic systems. Analysis of their functions and evolution requires the establishment of a new biological discipline, proposed to be called symbiogenetics, which provides a basis for fundamental and applied research of the genetic control over different (symbiotic and biocenotic) biotic interactions. In ecology and agrobiology, the approaches of symbiogenetics are indispensable for optimising the interactions between the plants and the beneficial microbes to be used in ecosystem management and in sustainable crop production in which hazardous fertilisers and pesticides should be replaced by environmentally friendly microbial inoculants.

Even after death the endophytic fungus of Schedonorus phoenix reduces the arbuscular mycorrhizas of other plants

1 Plants can host microbial communities whose integrated functions are often responsible for their success. Understanding mechanisms regulating such functions is thus a major goal in ecology. 2 Fungal endophytes of grasses, particularly of the genus Neotyphodium, have been reported to reduce colonization of their host plant by arbuscular mycorrhizal (AM) fungi. However, it is unclear which mechanism(s) may explain the effects produced by the endophyte and whether these effects are present in nature. 3 We used Schedonorus phoenix (syn. Lolium arundinaceum) plants that were endophyte-free or infected with one of two strains of Neotyphodium coenophialum known to produce different putative allelochemicals to test the hypotheses that (i) allelopathic effects of the endophyte reduce AM fungal spore germination; and (ii) the allelochemical compound(s) are leached into the soil even after the death of S. phoenix, where they reduce AM fungal colonization of other plants. 4 In a first experiment, aqueous extracts from the shoots of S. phoenix were applied onto spores of the AM fungus Glomus intraradices to test germination effects. Both endophyte strains reduced spore germination by approximately 10% relative to endophyte-free controls. 5 In a second experiment, we placed dried shoot material (‘thatch’) on the soil surface of pots containing Bromus inermis, which were either inoculated with G. intraradices or not. We watered the plants through the thatch, relying upon leaching to translocate potential allelochemicals to the soil. AM fungal colonization of B. inermis was significantly reduced when thatch was infected with the common strain, but not with AR542, compared to the endophyte-free thatch. Furthermore, the arbuscule : vesicle ratio was 11-fold smaller when thatch was infected with the common strain compared to endophyte-free thatch, suggesting that G. intraradices was stressed by the presence of common strain-leachate. 6 We observed situations whereby two ecologically widespread plant-microbe symbioses interact. Potential mechanisms may include allelopathic effects, although other factors are also possible, and leaching is a mode of entry of putative endophyte-induced AM fungal inhibitors in soil. Understanding these processes is important as they affect AM fungal communities which contribute to plant success and, consequentially, grassland ecosystem dynamics.

MOLECULAR STRATEGIES AND AGRONOMIC IMPACTS OF PLANT-MICROBE SYMBIOSES

The molecular mechanism of the agronomically important nutritional and defensive plantmicrobe symbioses are reviewed. These symbioses are based on the signaling interactions which result in the development of novel tissue/cellular structures and of extended metabolic capacities in the partners which improve greatly the adaptive potential of plants due to an increased tolerance to biotic or abiotic stresses. The molecular, genetic and ecological knowledge on plant-microbe interactions provide a strategy for a sustainable crop production based on substituting the agrochemicals (mineral fertilizers, pesticides) by the microbial inoculants. An improvement of plantmicrobe symbioses should involve the coordinated partners' modifications resulted in complementary combinations of their genotypes. The research presented in this paper is supported by grants from Russian Foundation of Basic Research (04-04-48457, 06-04-48800, 06-04-89000NWO); NWO Centre of Excellence: 047. 018. 001.