Plant-fungus symbiosis: One receptor to switch on the green light.

Arbuscular mycorrhiza, an ancient symbiosis with soil fungi, support mineral nutrition in most plants. How roots recognize such symbiotic fungi has long been debated. Recent research identifies a Medicago truncatula receptor as a key player in triggering symbiont accommodation responses.

Boosting species evenness, productivity and weed control in a mixed meadow by promoting arbuscular mycorrhizas.

Lowland meadows represent aboveground and belowground biodiversity reservoirs in intensive agricultural areas, improving water retention and filtration, ensuring forage production, contrasting erosion and contributing to soil fertility and carbon sequestration. Besides such major ecosystem services, the presence of functionally different plant species improves forage quality, nutritional value and productivity, also limiting the establishment of weeds and alien species. Here, we tested the effectiveness of a commercial seed mixture in restoring a lowland mixed meadow in the presence or absence of inoculation with arbuscular mycorrhizal (AM) fungi and biostimulation of symbiosis development with the addition of short chain chito-oligosaccharides (CO). Plant community composition, phenology and productivity were regularly monitored alongside AM colonization in control, inoculated and CO-treated inoculated plots. Our analyses revealed that the CO treatment accelerated symbiosis development significantly increasing root colonization by AM fungi. Moreover, the combination of AM fungal inoculation and CO treatment improved plant species evenness and productivity with more balanced composition in forage species. Altogether, our study presented a successful and scalable strategy for the reintroduction of mixed meadows as valuable sources of forage biomass; demonstrated the positive impact of CO treatment on AM development in an agronomic context, extending previous observations developed under controlled laboratory conditions and leading the way to the application in sustainable agricultural practices.

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.

Nonbinary fungal signals and calcium-mediated transduction in plant immunity and symbiosis.

Chitin oligomers (COs) are among the most common and active fungal elicitors of plant responses. Short-chain COs from symbiotic arbuscular mycorrhizal fungi activate accommodation responses in the host root, while long-chain COs from pathogenic fungi are acknowledged to trigger defence responses. The modulation of intracellular calcium concentration - a common second messenger in a wide variety of plant signal transduction processes - plays a central role in both signalling pathways with distinct signature features. Nevertheless, mounting evidence suggests that plant immunity and symbiosis signalling partially overlap at multiple levels. Here, we elaborate on recent findings on this topic, highlighting the nonbinary nature of chitin-based fungal signals, their perception and their interpretation through Ca2+ -mediated intracellular signals. Based on this, we propose that plant perception of symbiotic and pathogenic fungi is less clear-cut than previously described and involves a more complex scenario in which partially overlapping and blurred signalling mechanisms act upstream of the unambiguous regulation of gene expression driving accommodation or defence responses.

Quantifying root colonization by a symbiotic fungus using automated image segmentation and machine learning approaches.

Arbuscular mycorrhizas (AM) are one of the most widespread symbiosis on earth. This plant-fungus interaction involves around 72% of plant species, including most crops. AM symbiosis improves plant nutrition and tolerance to biotic and abiotic stresses. The fungus, in turn, receives carbon compounds derived from the plant photosynthetic process, such as sugars and lipids. Most studies investigating AM and their applications in agriculture requires a precise quantification of the intensity of plant colonization. At present, the majority of researchers in the field base AM quantification analyses on manual visual methods, prone to operator errors and limited reproducibility. Here we propose a novel semi-automated approach to quantify AM fungal root colonization based on digital image analysis comparing three methods: (i) manual quantification (ii) image thresholding, (iii) machine learning. We recognize machine learning as a very promising tool for accelerating, simplifying and standardizing critical steps in analysing AM quantification, answering to an urgent need by the scientific community studying this symbiosis.

Strigolactones promote the localization of the ABA exporter ABCG25 at the plasma membrane in root epidermal cells of Arabidopsis thaliana.

The phytohormones strigolactones crosstalk with abscisic acid (ABA) in acclimation to osmotic stress, as ascertained in leaves. However, our knowledge about underground tissues is limited, and lacking in Arabidopsis: whether strigolactones affect ABA transport across plasma membranes has never been addressed. We evaluated the effect of strigolactones on the localization of ATP BINDING CASSETTE G25 (ABCG25), an ABA exporter in Arabidopsis thaliana. Wild-type, strigolactone-insensitive, and strigolactone-depleted seedlings expressing a green fluorescent protein:ABCG25 construct were treated with ABA or strigolactones, and green fluorescent protein was quantified by confocal microscopy in different subcellular compartments of epidermal root cells. We show that strigolactones promote the localization of an ABA transporter at the plasma membrane by enhancing its endosomal recycling. Genotypes altered in strigolactone synthesis or perception are not impaired in ABCG25 recycling promotion by ABA, which acts downstream or independent of strigolactones in this respect. Additionally, we confirm that osmotic stress decreases strigolactone synthesis in A. thaliana root cells, and that this decrease may support local ABA retention under low water availability by allowing ABCG25 internalization. Thus, we propose a new mechanism for ABA homeostasis regulation in the context of osmotic stress acclimation: the fine-tuning by strigolactones of ABCG25 localization in root cells.

Long-lasting impact of chitooligosaccharide application on strigolactone biosynthesis and fungal accommodation promotes arbuscular mycorrhiza in Medicago truncatula.

The establishment of arbuscular mycorrhiza (AM) between plants and Glomeromycotina fungi is preceded by the exchange of chemical signals: fungal released Myc-factors, including chitooligosaccharides (CO) and lipo-chitooligosaccharides (LCO), activate plant symbiotic responses, while root-exuded strigolactones stimulate hyphal branching and boost CO release. Furthermore, fungal signaling reinforcement through CO application was shown to promote AM development in Medicago truncatula, but the cellular and molecular bases of this effect remained unclear. Here, we focused on long-term M. truncatula responses to CO treatment, demonstrating its impact on the transcriptome of both mycorrhizal and nonmycorrhizal roots over several weeks and providing an insight into the mechanistic bases of the CO-dependent promotion of AM colonization. CO treatment caused the long-lasting regulation of strigolactone biosynthesis and fungal accommodation-related genes. This was mirrored by an increase in root didehydro-orobanchol content, and the promotion of accommodation responses to AM fungi in root epidermal cells. Lastly, an advanced downregulation of AM symbiosis marker genes was observed at the latest time point in CO-treated plants, in line with an increased number of senescent arbuscules. Overall, CO treatment triggered molecular, metabolic, and cellular responses underpinning a protracted acceleration of AM development.

Extraction of short chain chitooligosaccharides from fungal biomass and their use as promoters of arbuscular mycorrhizal symbiosis.

Short chain chitooligosaccharides (COs) are chitin derivative molecules involved in plant-fungus signaling during arbuscular mycorrhizal (AM) interactions. In host plants, COs activate a symbiotic signalling pathway that regulates AM-related gene expression. Furthermore, exogenous CO application was shown to promote AM establishment, with a major interest for agricultural applications of AM fungi as biofertilizers. Currently, the main source of commercial COs is from the shrimp processing industry, but purification costs and environmental concerns limit the convenience of this approach. In an attempt to find a low cost and low impact alternative, this work aimed to isolate, characterize and test the bioactivity of COs from selected strains of phylogenetically distant filamentous fungi: Pleurotus ostreatus, Cunninghamella bertholletiae and Trichoderma viride. Our optimized protocol successfully isolated short chain COs from lyophilized fungal biomass. Fungal COs were more acetylated and displayed a higher biological activity compared to shrimp-derived COs, a feature that-alongside low production costs-opens promising perspectives for the large scale use of COs in agriculture.

Unique and common traits in mycorrhizal symbioses.

Mycorrhizas are among the most important biological interkingdom interactions, as they involve ~340,000 land plants and ~50,000 taxa of soil fungi. In these mutually beneficial interactions, fungi receive photosynthesis-derived carbon and provide the host plant with mineral nutrients such as phosphorus and nitrogen in exchange. More than 150 years of research on mycorrhizas has raised awareness of their biology, biodiversity and ecological impact. In this Review, we focus on recent phylogenomic, molecular and cell biology studies to present the current state of knowledge of the origin of mycorrhizal fungi and the evolutionary history of their relationship with land plants. As mycorrhizas feature a variety of phenotypes, depending on partner taxonomy, physiology and cellular interactions, we explore similarities and differences between mycorrhizal types. During evolution, mycorrhizal fungi have refined their biotrophic capabilities to take advantage of their hosts as food sources and protective niches, while plants have developed multiple strategies to accommodate diverse fungal symbionts. Intimate associations with pervasive ecological success have originated at the crossroads between these two evolutionary pathways. Our understanding of the biological processes underlying these symbioses, where fungi act as biofertilizers and bioprotectors, provides the tools to design biotechnological applications addressing environmental and agricultural challenges.

Fluorescent Staining of Arbuscular Mycorrhizal Structures Using Wheat Germ Agglutinin (WGA) and Propidium Iodide.

The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial.We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.

An endophytic Fusarium-legume association is partially dependent on the common symbiotic signalling pathway.

Legumes interact with a wide range of microbes in their root systems, ranging from beneficial symbionts to pathogens. Symbiotic rhizobia and arbuscular mycorrhizal glomeromycetes trigger a so-called common symbiotic signalling pathway (CSSP), including the induction of nuclear calcium spiking in the root epidermis. By combining gene expression analysis, mutant phenotypic screening and analysis of nuclear calcium elevations, we demonstrate that recognition of an endophytic Fusarium solani strain K (FsK) in model legumes is initiated via perception of chitooligosaccharidic molecules and is, at least partially, CSSP-dependent. FsK induced the expression of Lysin-motif receptors for chitin-based molecules, CSSP members and CSSP-dependent genes in Lotus japonicus. In LysM and CSSP mutant/RNAi lines, root penetration and fungal intraradical progression was either stimulated or limited, whereas FsK exudates triggered CSSP-dependent nuclear calcium spiking, in epidermal cells of Medicago truncatula root organ cultures. Our results corroborate CSSP being involved in the perception of signals from other microbes beyond the restricted group of symbiotic interactions sensu stricto.

Short chain chito-oligosaccharides promote arbuscular mycorrhizal colonization in Medicago truncatula.

During the establishment of arbuscular mycorrhizal (AM) symbiosis, the fungus and the host plant exchange chemical signals that are crucial to reciprocal recognition. Short-chain chitin oligomers (CO) released by AM fungi are known to trigger symbiotic signaling in all host plant species tested. Here we applied exogenous CO, derived from crustacean exoskeleton, to pot-grown Medicago truncatula inoculated with the AM fungus Funneliformis mosseae and investigated root colonization, plant gene regulation and biomass production. CO treatment strongly promoted AM colonization with significant increases in arbuscule development, biomass production and photosynthetic surface compared to untreated mycorrhizal plants. Gene expression analyses indicated that CO treatment anticipated the expression of MtBCP and MtPT4 plant symbiotic markers, during the first two weeks post inoculation. Altogether, our results provide evidence that plant treatment with symbiotic fungal elicitors, anticipated and enhanced AM development, encouraging the use of CO to promote AM establishment in sustainable agricultural practices.

Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis.

BACKGROUND: The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. RESULTS: We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. CONCLUSIONS: In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.

Colonization of legumes by an endophytic Fusarium solani strain FsK reveals common features to symbionts or pathogens.

Plant cellular responses to endophytic filamentous fungi are scarcely reported, with the majority of described colonization processes in plant-fungal interactions referring to either pathogens or true symbionts. Fusarium solani strain K (FsK) is a root endophyte of Solanum lycopersicum, which protects against root and foliar pathogens. Here, we investigate the association of FsK with two legumes (Lotus japonicus and Medicago truncatula) and report on colonization patterns and plant responses during the establishment of the interaction. L. japonicus plants colonized by FsK complete their life cycle and exhibit no apparent growth defects under normal conditions. We followed the growth of FsK within root-inoculated plants spatiotemporally and showed the capability of the endophyte to migrate to the stem. In a bipartite system comprising of the endophyte and either whole plants or root organ cultures, we studied the plant sub-cellular responses to FsK recognition, using optical, confocal and transmission electron microscopy. A polarized reorganization of the root cell occurs: endoplasmic reticulum/cytoplasm accumulation and nuclear placement at contact sites, occasional development of papillae underneath hyphopodia and membranous material rearrangements towards penetrating hyphae. Fungal hyphae proliferate within the vascular bundle of the plant. Plant cell death is involved in fungal colonization of the root. Our data suggest that the establishment of FsK within legume tissues requires fungal growth adaptations and plant cell-autonomous responses, known to occur during both symbiotic and pathogenic plant-fungal interactions. We highlight the overlooked plasticity of endophytic fungi upon plant colonization, and introduce a novel plant-endophyte association.