Nuclear factors NF-YC3 and NF-YBs positively regulate arbuscular mycorrhizal symbiosis in tomato.

The involvement of nuclear factor Y (NF-Y) in transcriptional reprogramming during arbuscular mycorrhizal symbiosis has been demonstrated in several plant species. However, a comprehensive picture is lacking. We showed that the spatial expression of NF-YC3 was observed in cortical cells containing arbuscules via the cis-regulatory element GCC boxes. Moreover, the NF-YC3 promoter was transactivated by the combination of CYCLOPS and autoactive calcium and calmodulin-dependent kinase (CCaMK) via GCC boxes. Knockdown of NF-YC3 significantly reduced the abundance of all intraradical fungal structures and affected arbuscule size. BCP1, SbtM1, and WRI5a, whose expression associated with NF-YC3 levels, might be downstream of NF-YC3. NF-YC3 interacted with NF-YB3a, NF-YB5c, or NF-YB3b, in yeast (Saccharomyces cerevisiae) and in planta, and interacted with NF-YA3a in yeast. Spatial expression of three NF-YBs was observed in all cell layers of roots under both mock and mycorrhizal conditions. Simultaneous knockdown of three NF-YBs, but not individually, reduced the fungal colonization level, suggesting that there might be functional redundancy of NF-YBs to regulate AM symbiosis. Collectively, our data suggest that NF-YC3 and NF-YBs positively regulate AM symbiosis in tomato, and arbuscule-related NF-YC3 may be an important downstream gene of the common symbiosis signaling pathway.

Joint evolution of mutualistic interactions, pollination, seed dispersal mutualism, and mycorrhizal symbiosis in trees.

Mycorrhizal symbiosis, seed dispersal, and pollination are recognized as the most prominent mutualistic interactions in terrestrial ecosystems. However, it remains unclear how these symbiotic relationships have interacted to contribute to current plant diversity. We analyzed evolutionary relationships among mycorrhizal type, seed dispersal mode, and pollination mode in two global databases of 699 (database I) and 10 475 (database II) tree species. Although database II had been estimated from phylogenetic patterns and therefore had lower certainty of the mycorrhizal type than database I, whose mycorrhizal type was determined by direct observation, database II allowed analysis of many more taxa from more regions than database I. We found evidence of joint evolution of all three features in both databases. This result is robust to the effects of both sampling bias and missing taxa. Most arbuscular mycorrhizal-associated trees had endozoochorous (biotic) seed dispersal and biotic pollination, with long dispersal distances, whereas most ectomycorrhizal-associated trees had anemochorous (abiotic) seed dispersal and wind (abiotic) pollination mode, with shorter dispersal distances. These results provide a novel scenario in mutualistic interactions, seed dispersal, pollination, and mycorrhizal symbiosis types, which have jointly evolved and shaped current tree diversity and forest ecosystem world-wide.

Domestication of rice may have changed its arbuscular mycorrhizal properties by modifying phosphorus nutrition-related traits and decreasing symbiotic compatibility.

Modern cultivated rice (Oryza sativa) typically experiences limited growth benefits from arbuscular mycorrhizal (AM) symbiosis. This could be due to the long-term domestication of rice under favorable phosphorus conditions. However, there is limited understanding of whether and how the rice domestication has modified AM properties. This study compared AM properties between a collection of wild (Oryza rufipogon) and domesticated rice genotypes and investigated the mechanisms underlying their differences by analyzing physiological, genomic, transcriptomic, and metabolomic traits critical for AM symbiosis. The results revealed significantly lower mycorrhizal growth responses and colonization intensity in domesticated rice compared to wild rice, and this change of AM properties may be associated with the domestication modifications of plant phosphorus utilization efficiency at physiological and genomic levels. Domestication also resulted in a decrease in the activity of the mycorrhizal phosphorus acquisition pathway, which may be attributed to reduced mycorrhizal compatibility of rice roots by enhancing defense responses like root lignification and reducing carbon supply to AM fungi. In conclusion, rice domestication may have changed its AM properties by modifying P nutrition-related traits and reducing symbiotic compatibility. This study offers new insights for improving AM properties in future rice breeding programs to enhance sustainable agricultural production.

Mycorrhization enhances plant growth and stabilizes biomass allocation under drought.

Plants and their symbionts, such as arbuscular mycorrhizal (AM) fungi, are increasingly subjected to various environmental stressors due to climate change, including drought. As a response to drought, plants generally allocate more biomass to roots over shoots, thereby facilitating water uptake. However, whether this biomass allocation shift is modulated by AM fungi remains unknown. Based on 5691 paired observations from 154 plant species, we conducted a meta-analysis to evaluate how AM fungi modulate the responses of plant growth and biomass allocation (e.g., root-to-shoot ratio, R/S) to drought. We found that AM fungi attenuate the negative impact of drought on plant growth, including biomass production, photosynthetic performance and resource (e.g. nutrient and water) uptake. Accordingly, drought significantly increased R/S in non-inoculated plants, but not in plants symbiotic with established AM fungal symbioses. These results suggest that AM fungi promote plant growth and stabilize their R/S through facilitating nutrient and water uptake in plants under drought. Our findings highlight the crucial role of AM fungi in enhancing plant resilience to drought by optimizing resource allocation. This knowledge opens avenues for sustainable agricultural practices that leverage symbiotic relationships for climate adaptation.

Arbuscular mycorrhizal fungi attenuate negative impact of drought on soil functions.

Although positive effects of arbuscular mycorrhizal (AM) fungi on plant performance under drought have been well documented, how AM fungi regulate soil functions and multifunctionality requires further investigation. In this study, we first performed a meta-analysis to test the potential role of AM fungi in maintaining soil functions under drought. Then, we conducted a greenhouse experiment, using a pair of hyphal ingrowth cores to spatially separate the growth of AM fungal hyphae and plant roots, to further investigate the effects of AM fungi on soil multifunctionality and its resistance against drought. Our meta-analysis showed that AM fungi promote multiple soil functions, including soil aggregation, microbial biomass and activities of soil enzymes related to nutrient cycling. The greenhouse experiment further demonstrated that AM fungi attenuate the negative impact of drought on these soil functions and thus multifunctionality, therefore, increasing their resistance against drought. Moreover, this buffering effect of AM fungi persists across different frequencies of water supply and plant species. These findings highlight the unique role of AM fungi in maintaining multiple soil functions by mitigating the negative impact of drought. Our study highlights the importance of AM fungi as a nature-based solution to sustaining multiple soil functions in a world where drought events are intensifying.

Measuring leaf and root functional traits uncovers multidimensionality of plant responses to arbuscular mycorrhizal fungi.

PREMISE: While many studies have measured the aboveground responses of plants to mycorrhizal fungi at a single time point, little is known about how plants respond belowground or across time to mycorrhizal symbiosis. By measuring belowground responses and growth over time in many plant species, we create a more complete picture of how mycorrhizal fungi benefit their hosts. METHODS: We grew 26 prairie plant species with and without mycorrhizal fungi and measured 14 functional traits to assess above- and belowground tissue quality and quantity responses and changes in resource allocation. We used function-valued trait (FVT) modeling to characterize changes in species growth rate when colonized. RESULTS: While aboveground biomass responses were positive, the response of traits belowground were much more variable. Changes in aboveground biomass accounted for 60.8% of the variation in mycorrhizal responses, supporting the use of aboveground biomass response as the primary response trait. Responses belowground were not associated with aboveground responses and accounted for 18.3% of the variation. Growth responses over time were highly variable across species. Interestingly, none of the measured responses were phylogenetically conserved. CONCLUSIONS: Mycorrhizal fungi increase plant growth in most scenarios, but the effects of these fungi belowground and across time are more complicated. This study highlights how differences in plant allocation priorities might affect how they utilize the benefits from mycorrhizal fungi. Identifying and characterizing these differences is a key step to understanding the effects of mycorrhizal mutualisms on whole plant physiology.

Arbuscular mycorrhizal fungi offset NH3 emissions in temperate meadow soil under simulated warming and nitrogen deposition.

Most soil ammonia (NH3) emissions originate from soil nitrogen (N) that has been in the form of exchangeable ammonium. Emitted NH3 not only induces nutrient loss but also has adverse effects on the cycling of N and accelerates global warming. There is evidence that arbuscular mycorrhizal (AM) fungi can alleviate N loss by reducing N2O emissions in N-limited ecosystems, however, some studies have also found that global changes, such as warming and N deposition, can affect the growth and development of AM fungi and alter their functionality. Up to now, the impact of AM fungi on NH3 emissions, and whether global changes reduce the AM fungi's contribution to NH3 emissions reduction, has remained unclear. In this study, we examined how warming, N addition, and AM fungi alter NH3 emissions from high pH saline soils typical of a temperate meadow through a controlled microscopic experiment. The results showed that warming significantly increased soil NH3 emissions, but N addition and combined warming plus N addition had no impact. Inoculations with AM fungi strongly reduced NH3 emissions both under warming and N addition, but AM fungi effects were more pronounced under warming than following N addition. Inoculation with AM fungi reduced soil NH4+-N content and soil pH, and increased plant N content and soil net N mineralization rate while increasing the abundance of ammonia-oxidizing bacterial (AOB) gene. Structural equation modeling (SEM) shows that the regulation of NH3 emissions by AM fungi may be related to soil NH4+-N content and soil pH. These findings highlight that AM fungi can reduce N loss in the form of NH3 by increasing N turnover and uptake under global changes; thus, AM fungi play a vital role in alleviating the aggravation of N loss caused by global changes and in mitigating environmental pollution in the future.

Spatial Structure within Root Systems Moderates Stability of Arbuscular Mycorrhizal Mutualism and Plant-Soil Feedbacks.

AbstractThe persistence of mutualisms is paradoxical, as there are fitness incentives for exploitation. This is particularly true for plant-microbe mutualisms like arbuscular mycorrhizae (AM), which are promiscuously horizontally transmitted. Preferential allocation by hosts to the best mutualist can stabilize horizontal mutualisms; however, preferential allocation is imperfect, with its fidelity likely depending on the spatial structure of symbionts in plant roots. In this study we tested AM mutualisms' dependence on two dimensions of spatial structure-the initial spatial association of fungi and the ease of fungal dispersal-through three complementary experiments. We found that fitness of the beneficial AM fungus increased when fungi were initially separate, while initial spatial mixing benefited the fitness of the nonbeneficial fungus. These effects were strongest when dispersal was limited and hosts could discriminate. Additionally, we found that changes in AM fungal proportional abundance induced by spatial structure in roots of a preferentially allocating host produced positive feedbacks on plant growth, showing that interactions between spatial structure and host choice can determine the direction of plant-soil feedbacks. Our results suggest that symbiont spatial structure within plant roots may act as an important modifier of plant preferential allocation and the dynamics of mycorrhizal mutualisms, with potentially cascading effects on plant-plant interactions.

Soil organic matter dynamics mediated by arbuscular mycorrhizal fungi - an updated conceptual framework.

Arbuscular mycorrhizal (AM) fungi play an important role in soil organic matter (SOM) formation and stabilization. Previous studies have emphasized organic compounds produced by AM fungi as persistent binding agents for aggregate formation and SOM storage. This concept overlooks the multiple biogeochemical processes mediated by AM fungal activities, which drive SOM generation, reprocessing, reorganization, and stabilization. Here, we propose an updated conceptual framework to facilitate a mechanistic understanding of the role of AM fungi in SOM dynamics. In this framework, four pathways for AM fungi-mediated SOM dynamics are included: 'Generating', AM fungal exudates and biomass serve as key sources of SOM chemodiversity; 'Reprocessing', hyphosphere microorganisms drive SOM decomposition and resynthesis; 'Reorganizing', AM fungi mediate soil physical changes and influence SOM transport, redistribution, transformation, and storage; and 'Stabilizing', AM fungi drive mineral weathering and organo-mineral interactions for SOM stabilization. Moreover, we discuss the AM fungal role in SOM dynamics at different scales, especially when translating results from small scales to complex larger scales. We believe that working with this conceptual framework can allow a better understanding of AM fungal role in SOM dynamics, therefore facilitating the development of mycorrhiza-based technologies toward soil health and global change mitigation.

Accumulation in nutrient acquisition strategies of arbuscular mycorrhizal fungi and plant roots in poor and heterogeneous soils of karst shrub ecosystems.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi and roots play important roles in plant nutrient acquisition, especially in nutrient poor and heterogeneous soils. However, whether an accumulation strategy of AM fungi and root exists in such soils of karst shrubland ecosystems remains unclear. Root traits related to nutrient acquisition (root biomass, AM colonisation, root acid phosphatase activity and N2 fixation) were measured in two N2-fixing plants (i.e. Albizia odoratissima (Linn. f.) Benth. and Cajanus cajan (Linn.) Millsp.) that were grown in heterogeneous or homogeneous nutrient (ammonium) soil with and without AM fungi inoculation. RESULTS: Both of these plants had higher AM colonisation, root biomass and relative growth rate (RGR), but lower N2 fixation and root acid phosphatase activity in the rhizosphere in the heterogeneous soil environment, than that in the homogeneous soil environment. Plants grown in the AM fungi-inoculated heterogeneous soil environment had increased root biomass and root acid phosphatase activity compared with those grown in soil without inoculation. AM colonisation was negatively correlated with the N2 fixation rate of A. odoratissima, while it was not significantly correlated with the root phosphatase activity. CONCLUSIONS: Our results indicated that enhanced AM symbiosis and root biomass increased the absorptive surfaces for nutrient acquisition, highlighting the accumulation strategies of AM and root traits for plant nutrient acquisition in nutrient poor and heterogeneous soils of the karst shrubland ecosystem.

Elevation and plant species identity jointly shape a diverse arbuscular mycorrhizal fungal community in the High Arctic.

Knowledge about the distribution and local diversity patterns of arbuscular mycorrhizal (AM) fungi are limited for extreme environments such as the Arctic, where most studies have focused on spore morphology or root colonization. We here studied the joint effects of plant species identity and elevation on AM fungal distribution and diversity. We sampled roots of 19 plant species in 18 locations in Northeast Greenland, using next generation sequencing to identify AM fungi. We studied the joint effect of plant species, elevation and selected abiotic conditions on AM fungal presence, richness and composition. We identified 29 AM fungal virtual taxa (VT), of which six represent putatively new VT. Arbuscular mycorrhizal fungal presence increased with elevation, and as vegetation cover and the active soil layer decreased. Arbuscular mycorrhizal fungal composition was shaped jointly by elevation and plant species identity. We demonstrate that the Arctic harbours a relatively species-rich and nonrandomly distributed diversity of AM fungi. Given the high diversity and general lack of knowledge exposed herein, we encourage further research into the diversity, drivers and functional role of AM fungi in the Arctic. Such insight is urgently needed for an area with some of the globally highest rates of climate change.

Arbuscular mycorrhizal symbiosis enhances tomato lateral root formation by modulating CEP2 peptide expression.

Plant lateral root (LR) growth usually is stimulated by arbuscular mycorrhizal (AM) symbiosis. However, the molecular mechanism is still unclear. We used gene expression analysis, peptide treatment and virus-induced gene alteration assays to demonstrate that C-terminally encoded peptide (CEP2) expression in tomato was downregulated during AM symbiosis to mitigate its negative effect on LR formation through an auxin-related pathway. We showed that enhanced LR density and downregulated CEP2 expression were observed during mycorrhizal symbiosis. Synthetic CEP2 peptide treatment reduced LR density and impaired the expression of genes involved in indole-3-butyric acid (IBA, the precursor of IAA) to IAA conversion, auxin polar transport and the LR-related signaling pathway; however, application of IBA or synthetic auxin 1-naphthaleneacetic acid (NAA) to the roots may rescue both defective LR formation and reduced gene expression. CEP receptor 1 (CEPR1) might be the receptor of CEP2 because its knockdown plants did not respond to CEP2 treatment. Most importantly, the LR density of CEP2 overexpression or knockdown plants could not be further increased by AM inoculation, suggesting that CEP2 was critical for AM-induced LR formation. These results indicated that AM symbiosis may regulate root development by modulating CEP2, which affects the auxin-related pathway.

A CCaMK/Cyclops response element in the promoter of Lotus japonicus calcium-binding protein 1 (CBP1) mediates transcriptional activation in root symbioses.

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein 1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter : reporter fusions to transactivation and spatio-temporal expression analysis and screened ethyl methanesulphonate (EMS)-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the calcium and calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

The mycorrhizal symbiosis alters the plant defence strategy in a model legume plant.

Arbuscular mycorrhizal (AM) symbiosis modulates plant-herbivore interactions. Still, how it shapes the overall plant defence strategy and the mechanisms involved remain unclear. We investigated how AM symbiosis simultaneously modulates plant resistance and tolerance to a shoot herbivore, and explored the underlying mechanisms. Bioassays with Medicago truncatula plants were used to study the effect of the AM fungus Rhizophagus irregularis on plant resistance and tolerance to Spodoptera exigua herbivory. By performing molecular and chemical analyses, we assessed the impact of AM symbiosis on herbivore-triggered phosphate (Pi)- and jasmonate (JA)-related responses. Upon herbivory, AM symbiosis led to an increased leaf Pi content by boosting the mycorrhizal Pi-uptake pathway. This enhanced both plant tolerance and herbivore performance. AM symbiosis counteracted the herbivore-triggered JA burst, reducing plant resistance. To disentangle the role of the mycorrhizal Pi-uptake pathway in the plant's response to herbivory, we used the mutant line ha1-2, impaired in the H+ -ATPase gene HA1, which is essential for Pi-uptake via the mycorrhizal pathway. We found that mycorrhiza-triggered enhancement of herbivore performance was compromised in ha1-2 plants. AM symbiosis thus affects the defence pattern of M. truncatula by altering resistance and tolerance simultaneously. We propose that the mycorrhizal Pi-uptake pathway is involved in the modulation of the plant defence strategy.

Soil fungal networks moderate density-dependent survival and growth of seedlings.

Pathogenic and mutualistic fungi have contrasting effects on seedling establishment, but it remains unclear whether density-dependent survival and growth are regulated by access to different types of mycorrhizal fungal networks supported by neighbouring adult trees. Here, we conducted an extensive field survey to test how mycorrhizal and pathogenic fungal colonization of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) seedlings in a subtropical forest respond to density of neighbouring adult trees. In addition, we undertook a hyphal exclusion experiment to explicitly test the role of soil fungal networks in driving density-dependent effects on seedling growth and survival. Conspecific adult density was a strong predictor for the relative abundance of putative pathogens, which was greater in roots of AM than of ECM seedlings, while mycorrhizal fungal abundance and colonization were not consistently affected by conspecific adult density. Both ECM and AM fungal networks counteracted conspecific density-dependent mortality, but ECM fungi were more effective at weakening the negative effects of high seedling density than AM fungi. Our findings reveal a critical role of common fungal networks in mitigating negative density-dependent effects of pathogenic fungi on seedling establishment, which provides mechanistic insights into how soil fungal diversity shapes plant community structure in subtropical forests.

Mycorrhizal and environmental controls over root trait-decomposition linkage of woody trees.

Traits are critical in predicting decomposition that fuels carbon and nutrient cycling in ecosystems. However, our understanding of root trait-decomposition linkage, and especially its dependence on mycorrhizal type and environmental context, remains limited. We explored the control of morphological and chemical (carbon- and nutrient-related) traits over decomposition of absorptive roots in 30 tree species associated with either arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi in temperate and subtropical forests in China. Carbon-related traits (acid-unhydrolysable residue (AUR) and cellulose concentrations) had predominant control of root decomposition in AM species while nutrient-related traits (magnesium concentration) predominately controlled that in ECM species. Thicker absorptive roots decomposed faster in AM species as a result of their lower AUR concentrations, but more slowly in ECM angiosperm species potentially as a result of their higher magnesium concentrations. Root decomposition was linked to root nutrient economy in both forests while root diameter-decomposition coordination emerged only in the subtropical forest where root diameter and decomposition presented similar cross-species variations. Our findings suggest that root trait-decomposition linkages differ strongly with mycorrhizal type and environment, and that root diameter can predict decomposition but in opposing directions and with contrasting mechanisms for AM and ECM species.

Tree growth response to shifting soil nutrient economy depends on mycorrhizal associations.

The mycorrhizal-associated nutrient economy hypothesis proposes a strong connection between plant and fungal traits and the dominant form of soil nutrients. If true, then shifting from an organic to an inorganic nutrient economy should benefit arbuscular mycorrhizal (AM) trees because they are more suited to acquiring inorganic forms of nutrients and have limited decomposing capabilities when compared with ectomycorrhizal (ECM) trees. An inorganic nutrient economy was experimentally promoted by applying inorganic phosphorus (P) fertiliser and/or elevating soil pH with lime in three Allegheny Plateau mixed-mesophytic forests. Trees were measured over seven growing seasons to determine how growth responded to the treatments based on mycorrhizal association. AM-associated trees showed increased growth in response to increased inorganic nutrients, but ECM tree growth was suppressed when compared with the control. We also observed that understory and mid-story trees responded to the treatments, but large overstory trees showed no significant growth response. Results support the hypothesis that AM trees respond positively to an inorganic nutrient economy. While raising pH in acidic soils can be detrimental to ECM tree growth, the exact mechanism for this response is unclear.

Growth and Photosynthetic Activity of Selected Spelt Varieties (Triticum aestivum ssp. spelta L.) Cultivated under Drought Conditions with Different Endophytic Core Microbiomes.

The role of the microbiome in the root zone is critically important for plants. However, the mechanism by which plants can adapt to environmental constraints, especially water deficit, has not been fully investigated to date, while the endophytic core microbiome of the roots of spelt (Triticum aestivum ssp. spelta L.) grown under drought conditions has received little attention. In this study, we hypothesize that differences in the endophytic core of spelt and common wheat root microbiomes can explain the variations in the growth and photosynthetic activity of those plants, especially under drought conditions. Our greenhouse experimental design was completely randomized in a 2 × 4 × 3 factorial scheme: two water regime levels (well-watered and drought), three spelt varieties (T. aestivum ssp. spelta L.: 'Badenstern', 'Badenkrone' and 'Zollernspelz' and one wheat variety: T. aestivum ssp. vulgare L: 'Dakotana') and three mycorrhizal levels (autoclaved soil inoculation with Rhizophagus irregularis, control (autoclaved soil) and natural inoculation (non-autoclaved soil-microorganisms from the field). During the imposed stress period, relative water content (RWC), leaf chlorophyll fluorescence, gas exchange and water use efficiency (WUE) were measured. Microscopic observations of the root surface through fungi isolation and identification were conducted. Our results indicate that 'Badenstern' was the most drought tolerant variety, followed by 'Zollernspelz' and 'Badenkrone,' while the common wheat variety 'Dakotana' was the most drought sensitive. Inoculation of 'Badenstern' with the mycorrhizal fungi R. irregularis contributed to better growth performance as evidenced by increased whole plant and stalk dry matter accumulation, as well as greater root length and volume. Inoculation of 'Zollernspelz' with arbuscular mycorrhizal fungi (AMF) enhanced the photochemical efficiency of Photosystem II and significantly improved root growth under drought conditions, which was confirmed by enhanced aboveground biomass, root dry weight and length. This study provides evidence that AMF have the potential to be beneficial for plant growth and dry matter accumulation in spelt varieties grown under drought conditions.

Dysfunction in the arbuscular mycorrhizal symbiosis has consistent but small effects on the establishment of the fungal microbiota in Lotus japonicus.

Most land plants establish mutualistic interactions with arbuscular mycorrhizal (AM) fungi. Intracellular accommodation of AM fungal symbionts remodels important host traits like root morphology and nutrient acquisition. How mycorrhizal colonization impacts plant microbiota is unclear. To understand the impact of AM symbiosis on fungal microbiota, ten Lotus japonicus mutants impaired at different stages of AM formation were grown in non-sterile natural soil and their root-associated fungal communities were studied. Plant mutants lacking the capacity to form mature arbuscules (arb- ) exhibited limited growth performance associated with altered phosphorus (P) acquisition and reduction-oxidation (redox) processes. Furthermore, arb- plants assembled moderately but consistently different root-associated fungal microbiota, characterized by the depletion of Glomeromycota and the concomitant enrichment of Ascomycota, including Dactylonectria torresensis. Single and co-inoculation experiments showed a strong reduction of root colonization by D. torresensis in the presence of AM fungus Rhizophagus irregularis, particularly in arbuscule-forming plants. Our results suggest that impairment of central symbiotic functions in AM host plants leads to specific changes in root microbiomes and in tripartite interactions between the host plant, AM and non-AM fungi. This lays the foundation for mechanistic studies on microbe-microbe and microbe-host interactions in AM symbiosis of the model L. japonicus.

Accumulation of phosphoinositides in distinct regions of the periarbuscular membrane.

Phosphoinositides and phosphatidic acid are small anionic lipids that comprise a minor proportion of total membrane lipids in eukaryotic cells but influence a broad range of cellular processes including endomembrane trafficking, signaling, exocytosis and endocytosis. To investigate the spatial distribution of phosphoinositides during arbuscular mycorrhizal symbiosis, we generated fluorescent reporters of PI(4,5)P2 and PI4P, as well as phosphatidic acid and diacylglycerol and used them to monitor lipid distribution on the cytoplasmic side of membrane bilayers in colonized cortical cells. The PI4P reporter accumulated strongly on the periarbuscular membrane (PAM) and transiently labeled Golgi bodies, while the PA reporter showed differential labeling of endomembranes and the PAM. Surprisingly, the PI(4,5)P2 reporter accumulated in small, discrete regions of the PAM on the arbuscule trunks, frequently in two regions on opposing sides of the hypha. A mutant reporter with reduced PI(4,5)P2 binding capacity did not show these accumulations. The PI(4,5)P2 -rich regions were detected at all phases of arbuscule development following branching, co-localized with membrane marker proteins potentially indicating high membrane bilayer content, and were associated with an alteration in morphology of the hypha. A possible analogy to the biotrophic interfacial membrane complex formed in rice infected with Magnaporthe orzyae is discussed.