Potential Regulatory Effects of Arbuscular Mycorrhizal Fungi on Lipid Metabolism of Maize in Response to Low-Temperature Stress.

The specific mechanisms underlying membrane lipid remodeling and changes in gene expression induced by arbuscular mycorrhizal fungi (AMF) in low-temperature-stressed plants are still unclear. In this study, physiological, transcriptomic, and lipidomic analyses were used to elucidate the physiological mechanisms by which AMF can enhance the adaptation of maize plants to low-temperature stress. The results showed that the relative electrical conductivity and malondialdehyde content of maize leaves were decreased after the inoculation with AMF, indicating that AMF reduced the peroxidation of membrane lipids and maintained the fluidity of the cell membrane. Transcriptomic analysis showed the presence of 702 differentially expressed genes induced by AMF in maize plants exposed to low-temperature stress. Furthermore, lipidomic analysis revealed changes in 10 lipid classes in AMF-inoculated maize plants compared with their noninoculated counterparts under low-temperature stress conditions. Lipid remodeling is an important strategy that arbuscular mycorrhizal plants adopt to cope with low-temperature stress.

Plant Immunity Modulation in Arbuscular Mycorrhizal Symbiosis and Its Impact on Pathogens and Pests.

Arbuscular mycorrhizal (AM) symbiosis is the oldest and most widespread mutualistic association on Earth and involves plants and soil fungi belonging to Glomeromycotina. A complex molecular, cellular, and genetic developmental program enables partner recognition, fungal accommodation in plant tissues, and activation of symbiotic functions such as transfer of phosphorus in exchange for carbohydrates and lipids. AM fungi, as ancient obligate biotrophs, have evolved strategies to circumvent plant defense responses to guarantee an intimate and long-lasting mutualism. They are among those root-associated microorganisms able to boost plants' ability to cope with biotic stresses leading to mycorrhiza-induced resistance (MIR), which can be effective across diverse hosts and against different attackers. Here, we examine the molecular mechanisms underlying the modulation of plant immunity during colonization by AM fungi and at the onset and display of MIR against belowground and aboveground pests and pathogens. Understanding the MIR efficiency spectrum and its regulation is of great importance to optimizing the biotechnological application of these beneficial microbes for sustainable crop protection.

Pathogenic nematodes exploit Achilles' heel of plant symbioses.

Cyst nematode parasites disrupt beneficial associations of crops with rhizobia and mycorrhiza. Chen et al. discovered the mechanism and demonstrated that the soybean cyst nematode Heterodera glycines secretes a chitinase that destroys key symbiotic signals from the microbial symbionts. The authors further developed a chitinase inhibitor that alleviates symbiosis inhibition.

Breaking barriers: improving time and space resolution of arbuscular mycorrhizal symbiosis with single-cell sequencing approaches.

The cell and molecular bases of arbuscular mycorrhizal (AM) symbiosis, a crucial plant-fungal interaction for nutrient acquisition, have been extensively investigated by coupling traditional RNA sequencing techniques of roots sampled in bulk, with methods to capture subsets of cells such as laser microdissection. These approaches have revealed central regulators of this complex relationship, yet the requisite level of detail to effectively untangle the intricacies of temporal and spatial development remains elusive.The recent adoption of single-cell RNA sequencing (scRNA-seq) techniques in plant research is revolutionizing our ability to dissect the intricate transcriptional profiles of plant-microbe interactions, offering unparalleled insights into the diversity and dynamics of individual cells during symbiosis. The isolation of plant cells is particularly challenging due to the presence of cell walls, leading plant researchers to widely adopt nuclei isolation methods. Despite the increased resolution that single-cell analyses offer, it also comes at the cost of spatial perspective, hence, it is necessary the integration of these approaches with spatial transcriptomics to obtain a comprehensive overview.To date, few single-cell studies on plant-microbe interactions have been published, most of which provide high-resolution cell atlases that will become crucial for fully deciphering symbiotic interactions and addressing future questions. In AM symbiosis research, key processes such as the mutual recognition of partners during arbuscule development within cortical cells, or arbuscule senescence and degeneration, remain poorly understood, and these advancements are expected to shed light on these processes and contribute to a deeper understanding of this plant-fungal interaction.

The receptor-like kinase ARK controls symbiotic balance across land plants.

The mutualistic arbuscular mycorrhizal (AM) symbiosis arose in land plants more than 450 million years ago and is still widely found in all major land plant lineages. Despite its broad taxonomic distribution, little is known about the molecular components underpinning symbiosis outside of flowering plants. The ARBUSCULAR RECEPTOR-LIKE KINASE (ARK) is required for sustaining AM symbiosis in distantly related angiosperms. Here, we demonstrate that ARK has an equivalent role in symbiosis maintenance in the bryophyte Marchantia paleacea and is part of a broad AM genetic program conserved among land plants. In addition, our comparative transcriptome analysis identified evolutionarily conserved expression patterns for several genes in the core symbiotic program required for presymbiotic signaling, intracellular colonization, and nutrient exchange. This study provides insights into the molecular pathways that consistently associate with AM symbiosis across land plants and identifies an ancestral role for ARK in governing symbiotic balance.

Genetic and functional traits limit the success of colonisation by arbuscular mycorrhizal fungi in a tomato wild relative.

To understand whether domestication had an impact on susceptibility and responsiveness to arbuscular mycorrhizal fungi (AMF) in tomato (Solanum lycopersicum), we investigated two tomato cultivars ("M82" and "Moneymaker") and a panel of wild relatives including S. neorickii, S. habrochaites and S. pennellii encompassing the whole Lycopersicon clade. Most genotypes revealed good AM colonisation levels when inoculated with the AMF Funneliformis mosseae. By contrast, both S. pennellii accessions analysed showed a very low colonisation, but with normal arbuscule morphology, and a negative response in terms of root and shoot biomass. This behaviour was independent of fungal identity and environmental conditions. Genomic and transcriptomic analyses revealed in S. pennellii the lack of genes identified within QTLs for AM colonisation, a limited transcriptional reprogramming upon mycorrhization and a differential regulation of strigolactones and AM-related genes compared to tomato. Donor plants experiments indicated that the AMF could represent a cost for S. pennellii: F. mosseae could extensively colonise the root only when it was part of a mycorrhizal network, but a higher mycorrhization led to a higher inhibition of plant growth. These results suggest that genetics and functional traits of S. pennellii are responsible for the limited extent of AMF colonisation.

Arbuscular mycorrhizal symbiosis with Rhizophagus irregularis DAOM197198 modifies the root transcriptome of walnut trees.

Walnut trees are cultivated and exploited worldwide for commercial timber and nut production. They are heterografted plants, with the rootstock selected to grow in different soil types and conditions and to provide the best anchorage, vigor, and resistance or tolerance to soil borne pests and diseases. However, no individual rootstock is tolerant of all factors that impact walnut production. In Europe, Juglans regia is mainly used as a rootstock. Like most terrestrial plants, walnut trees form arbuscular mycorrhizal symbioses, improving water and nutrient uptake and providing additional ecosystem services. Effects of arbuscular mycorrhizal symbiosis on root gene regulation, however, has never been assessed. We analyzed the response of one rootstock of J. regia to colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM197198. Plant growth as well as the nitrogen and phosphorus concentrations in roots and shoots were significantly increased in mycorrhizal plants versus non-colonized plants. In addition, we have shown that 1,549 genes were differentially expressed, with 832 and 717 genes up- and down-regulated, respectively. The analysis also revealed that some rootstock genes involved in plant nutrition through the mycorrhizal pathway, are regulated similarly as in other mycorrhizal woody species: Vitis vinifera and Populus trichocarpa. In addition, an enrichment analysis performed on GO and KEGG pathways revealed some regulation specific to J. regia (i.e., the juglone pathway). This analysis reinforces the role of arbuscular mycorrhizal symbiosis on root gene regulation and on the need to finely study the effects of diverse arbuscular mycorrhizal fungi on root gene regulation, but also of the scion on the functioning of an arbuscular mycorrhizal fungus in heterografted plants such as walnut tree.

Independent regulation of strigolactones and blumenols during arbuscular mycorrhizal symbiosis in rice.

The apocarotenoid strigolactones (SLs) facilitate pre-symbiotic communication between arbuscular mycorrhizal (AM) fungi and plants. Related blumenol-C-glucosides (blumenols), have also been associated with symbiosis, but the cues that are involved in the regulation of blumenol accumulation during AM symbiosis remain unclear. In rice, our analyses demonstrated a strict correlation between foliar blumenol abundance and intraradical fungal colonisation. More specifically, rice mutants affected at distinct stages of the interaction revealed that fungal cortex invasion was required for foliar blumenol accumulation. Plant phosphate status and D14L hormone signalling had no effect, contrasting their known role in induction of SLs. This a proportion of the SL biosynthetic enzymes, D27 and D17, are equally required for blumenol production. These results importantly clarify that, while there is a partially shared biosynthetic pathway between SL and blumenols, the dedicated induction of the related apocarotenoids occurs in response to cues acting at distinct stages during the root colonisation process. However, we reveal that neither SLs nor blumenols are essential for fungal invasion of rice roots.

The AMSlide for noninvasive time-lapse imaging of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis, the nutritional partnership between AM fungi and most plant species, is globally ubiquitous and of great ecological and agricultural importance. Studying the processes of AM symbiosis is confounded by its highly spatiotemporally dynamic nature. While microscopy methods exist to probe the spatial side of this plant-fungal interaction, the temporal side remains more challenging, as reliable deep-tissue time-lapse imaging requires both symbiotic partners to remain undisturbed over prolonged time periods. Here, we introduce the AMSlide: a noninvasive, high-resolution, live-imaging system optimised for AM symbiosis research. We demonstrate the AMSlide's applications in confocal microscopy of mycorrhizal roots, from whole colonisation zones to subcellular structures, over timeframes from minutes to weeks. The AMSlide's versatility for different microscope set-ups, imaging techniques, and plant and fungal species is also outlined. It is hoped that the AMSlide will be applied in future research to fill in the temporal blanks in our understanding of AM symbiosis, as well as broader root and rhizosphere processes.

Exploring the potential role of four Rhizophagus irregularis nuclear effectors: opportunities and technical limitations.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that interact with the roots of most land plants. The genome of the AMF model species Rhizophagus irregularis contains hundreds of predicted small effector proteins that are secreted extracellularly but also into the plant cells to suppress plant immunity and modify plant physiology to establish a niche for growth. Here, we investigated the role of four nuclear-localized putative effectors, i.e., GLOIN707, GLOIN781, GLOIN261, and RiSP749, in mycorrhization and plant growth. We initially intended to execute the functional studies in Solanum lycopersicum, a host plant of economic interest not previously used for AMF effector biology, but extended our studies to the model host Medicago truncatula as well as the non-host Arabidopsis thaliana because of the technical advantages of working with these models. Furthermore, for three effectors, the implementation of reverse genetic tools, yeast two-hybrid screening and whole-genome transcriptome analysis revealed potential host plant nuclear targets and the downstream triggered transcriptional responses. We identified and validated a host protein interactors participating in mycorrhization in the host.S. lycopersicum and demonstrated by transcriptomics the effectors possible involvement in different molecular processes, i.e., the regulation of DNA replication, methylglyoxal detoxification, and RNA splicing. We conclude that R. irregularis nuclear-localized effector proteins may act on different pathways to modulate symbiosis and plant physiology and discuss the pros and cons of the tools used.

Regulation of nitric oxide by phytoglobins in Lotus japonicus is involved in mycorrhizal symbiosis with Rhizophagus irregularis.

Various reactive molecular species are generated in plant-microbe interactions, and these species participate in defense and symbiotic responses. Leguminous plants successfully establish symbiosis by maintaining an appropriate level of nitric oxide (NO), which is generated in the roots and nodules during root nodule symbiosis. Phytoglobin (plant hemoglobin) controls NO levels in plants. In this study, we investigated mycorrhizal symbiosis, which occurs in more than 80% of land plants, between Rhizophagus irregularis and Lotus japonicus to clarify the involvement of phytoglobin-mediated NO regulation. The mycorrhizae of L. japonicus exhibited higher NO levels in the presence of R. irregularis than in its absence, especially at the infection site. LjGlb1-1, a phytoglobin that regulates NO level in L. japonicus, was upregulated during symbiosis with R. irregularis. In transformed hairy roots carrying the ProLjGlb1-1:GUS construct, LjGlb1-1 expression was observed at the R. irregularis infection site. We further examined the symbiotic phenotypes of L. japonicus lines with high and low LjGlb1-1 expression with R. irregularis. During mycorrhizal symbiosis, the high LjGlb1-1 expression line exhibited better growth than the wild-type, whereas the low expression line exhibited poor growth. In addition, the expression of LjPT4, a phosphate transporter specific to mycorrhizal symbiosis, was higher in the high LjGlb1-1 expression line, whereas that of the tubulin gene of R. irregularis was lower in the low LjGlb1-1 expression line than in the wild-type. These results confirm that NO regulation by LjGlb1-1 is involved in mycorrhizal symbiosis in L. japonicus, as it is reportedly in nitrogen-fixing symbiosis.

Integration of rice apocarotenoid profile and expression pattern of Carotenoid Cleavage Dioxygenases reveals a positive effect of ß-ionone on mycorrhization.

Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel reliable markers at both local and systemic levels and indicating a promoting role of ß-ionone in AM symbiosis establishment.

Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.

Comparative proteomic analysis identifies proteins associated with arbuscular mycorrhizal symbiosis in Poncirus trifoliata.

Citrus, one of the most widely cultivated fruit crops in the world, relies on arbuscular mycorrhizal fungi (AMF) to absorb nutrients and water from soil. However, the molecular mechanism of AM symbiosis (AMS) in citrus in general have largely been understudied. Here, using a TMT labeling proteomic approach, we identified 365 differentially expressed proteins (DEPs) in roots of Poncirus trifoliata (a common citrus rootstock) upon Rhizophagus irregularis colonization as compared with uninoculated roots, of which 287 were up-regulated and 78 were down-regulated. GO analysis revealed that the DEPs were mainly involved in biological processes such as negative regulation of endopeptidase inhibitor activity, negative regulation of endopeptidase, one-carbon metabolic process and carbohydrate metabolic process. KEGG enrichment analysis indicated that the DEPs were mainly involved in regulating metabolic pathways such as fatty acid biosynthesis, phenylpropanoid biosynthesis and carbon metabolism. Furthermore, 194 of the 365 DEPs were found to be associated with AMS-responsive genes by association analysis with our previous transcriptomes data, which highlighted the important roles of these proteins in AMS. One of the 194 DEPs, neutral ceramidase (PtNCER), was further chosen for function analysis via RNAi interfering its homologous gene MtNCER in a mycorrhizal model plant Medicago truncatula, which confirmed a positive role of NCER in AM establishment. Our results provided basic data and key candidate genes for genetic improvement of efficient nutrient uptake through AM establishment in citrus and other crops.

A journey into the world of small RNAs in the arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) symbiosis is a mutualistic interaction between fungi and most land plants that is underpinned by a bidirectional exchange of nutrients. AM development is a tightly regulated process that encompasses molecular communication for reciprocal recognition, fungal accommodation in root tissues and activation of symbiotic function. As such, a complex network of transcriptional regulation and molecular signaling underlies the cellular and metabolic reprogramming of host cells upon AM fungal colonization. In addition to transcription factors, small RNAs (sRNAs) are emerging as important regulators embedded in the gene network that orchestrates AM development. In addition to controlling cell-autonomous processes, plant sRNAs also function as mobile signals capable of moving to different organs and even to different plants or organisms that interact with plants. AM fungi also produce sRNAs; however, their function in the AM symbiosis remains largely unknown. Here, we discuss the contribution of host sRNAs in the development of AM symbiosis by considering their role in the transcriptional reprogramming of AM fungal colonized cells. We also describe the characteristics of AM fungal-derived sRNAs and emerging evidence for the bidirectional transfer of functional sRNAs between the two partners to mutually modulate gene expression and control the symbiosis.

A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation.

BACKGROUND: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Microbe-dependent and independent nitrogen and phosphate acquisition and regulation in plants.

Nitrogen (N) and phosphorus (P) are the most important macronutrients required for plant growth and development. To cope with the limited and uneven distribution of N and P in complicated soil environments, plants have evolved intricate molecular strategies to improve nutrient acquisition that involve adaptive root development, production of root exudates, and the assistance of microbes. Recently, great advances have been made in understanding the regulation of N and P uptake and utilization and how plants balance the direct uptake of nutrients from the soil with the nutrient acquisition from beneficial microbes such as arbuscular mycorrhiza. Here, we summarize the major advances in these areas and highlight plant responses to changes in nutrient availability in the external environment through local and systemic signals.