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Yield and grain characteristics of maize plants grown in open-field conditions were evaluated after inoculation with Micosat F® on two different soils under dry and watered conditions. The mycorrhizal frequency and intensity were higher in inoculated maize (87.8 vs 80.3% and 26.8 vs 17.5%, respectively). The abundance of arbuscules in the root system was also higher in inoculated plants (9.7 vs 5.8%). The treatment did not affect grain yield. Positive effect of Typic Eutrudept soil on grain yield was observed. The irrigation effect on grain yield was evident only under draught conditions. Chemical characteristics of grain did not change substantially according to the experimental treatments; nevertheless, the NIRs indicated some physical differences among mycorrhized and not mycorrhized samples. [ABSTRACT FROM AUTHOR]

Coffee is one of the most traded commodities world-wide. As with 70% of land plants, coffee is associated with arbuscular mycorrhizal (AM) fungi, but the molecular bases of this interaction are unknown. We studied the mycorrhizal phenotype of two commercially important Coffea arabica cultivars ('Typica National' and 'Catimor Amarillo'), upon Funnelliformis mosseae colonisation grown under phosphorus limitation, using an integrated functional approach based on multi-omics, physiology and biochemistry. The two cultivars revealed a strong biomass increase upon mycorrhization, even at low level of fungal colonisation, improving photosynthetic efficiency and plant nutrition. The more important iconic markers of AM symbiosis were activated: We detected two gene copies of AM-inducible phosphate (Pt4), ammonium (AM2) and nitrate (NPF4.5) transporters, which were identified as belonging to the C. arabica parental species (C. canephora and C. eugenioides) with both copies being upregulated. Transcriptomics data were confirmed by ions and metabolomics analyses, which highlighted an increased amount of glucose, fructose and flavonoid glycosides. In conclusion, both coffee cultivars revealed a high responsiveness to the AM fungus along their root-shoot axis, showing a clear-cut re-organisation of the major metabolic pathways, which involve nutrient acquisition, carbon fixation, and primary and secondary metabolism., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Two-Spotted Spider Mites (TSSMs, Tetranychus urticae Koch 1836 (Acari: Tetranychidae)) is one of the most important pests in many crop plants, and their feeding activity is based on sucking leaf cell contents. The purpose of this study was to evaluate the interaction between TSSMs and their host Lima bean ( Phaseolus lunatus ) by analyzing the metabolomics of leaf pigments and the transcriptomics of TSSM guanine production. We also used epifluorescence, confocal laser scanning, and transmission electron microscopies to study the morphology and structure of TSSMs and their excreta. Finally, we evaluated the potential photosynthetic ability of TSSMs and the activity and content of Ribulose-1,5-bisphosphate Carboxylase/Oxigenase (RubisCO). We found that TSSMs express several genes involved in guanine production, including Guanosine Monophosphate Synthetase ( GMPS ) and decoyinine (DCY), a potential inhibitor of GMPS, was found to reduce TSSMs proliferation in infested Lima bean leaves. Despite the presence of intact chloroplasts and chlorophyll in TSSMs, we demonstrate that TSSMs do not retain any photosynthetic activity. Our results show for the first time the transcriptomics of guanine production in TSSMs and provide new insight into the catabolic activity of TSSMs on leaf chlorophyll and carotenoids. Finally, we preliminary demonstrate that DCY has an acaricidal potential against TSSMs.

Durum wheat is one of the most important agricultural crops, currently providing 18% of the daily intake of calories and 20% of daily protein intake for humans. However, being wheat that is cultivated in arid and semiarid areas, its productivity is threatened by drought stress, which is being exacerbated by climate change. Therefore, the identification of drought tolerant wheat genotypes is critical for increasing grain yield and also improving the capability of crops to uptake and assimilate nutrients, which are seriously affected by drought. This work aimed to determine the effect of arbuscular mycorrhizal fungi (AMF) on plant growth under normal and limited water availability in two durum wheat genotypes (Svevo and Etrusco). Furthermore, we investigated how the plant nutritional status responds to drought stress. We found that the response of Svevo and Etrusco to drought stress was differentially affected by AMF. Interestingly, we revealed that AMF positively affected sulfur homeostasis under drought conditions, mainly in the Svevo cultivar. The results provide a valuable indication that the identification of drought tolerant plants cannot ignore their nutrient use efficiency or the impact of other biotic soil components (i.e., AMF).

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The apocarotenoid zaxinone promotes growth and suppresses strigolactone biosynthesis in rice. To shed light on the mechanisms underlying its growth-promoting effect, we employed a combined omics approach integrating transcriptomics and metabolomics analysis of rice seedlings treated with zaxinone, and determined the resulting changes at the cellular and hormonal levels. Metabolites as well as transcripts analysis demonstrate that zaxinone application increased sugar content and triggered glycolysis, the tricarboxylic acid cycle and other sugar-related metabolic processes in rice roots. In addition, zaxinone treatment led to an increased root starch content and induced glycosylation of cytokinins. The transcriptomic, metabolic and hormonal changes were accompanied by striking alterations of roots at cellular level, which showed an increase in apex length, diameter, and the number of cells and cortex cell layers. Remarkably, zaxinone did not affect the metabolism of roots in a strigolactone deficient mutant, suggesting an essential role of strigolactone in the zaxinone growth-promoting activity. Taken together, our results unravel zaxinone as a global regulator of the transcriptome and metabolome, as well as of hormonal and cellular composition of rice roots. Moreover, they suggest that zaxinone promotes rice growth most likely by increasing sugar uptake and metabolism, and reinforce the potential of this compound in increasing rice performance., (© 2021. The Author(s).)

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Zaxinone is an apocarotenoid regulatory metabolite required for normal rice growth and development. In addition, zaxinone has a large application potential in agriculture, due to its growth-promoting activity and capability to alleviate infestation by the root parasitic plant Striga through decreasing strigolactone (SL) production. However, zaxinone is poorly accessible to the scientific community because of its laborious organic synthesis that impedes its further investigation and utilization. In this study, we developed easy-to-synthesize and highly efficient mimics of zaxinone (MiZax). We performed a structure-activity relationship study using a series of apocarotenoids distinguished from zaxinone by different structural features. Using the obtained results, we designed several phenyl-based compounds synthesized with a high-yield through a simple method. Activity tests showed that MiZax3 and MiZax5 exert zaxinone activity in rescuing root growth of a zaxinone-deficient rice mutant, promoting growth, and reducing SL content in roots and root exudates of wild-type plants. Moreover, these compounds were at least as efficient as zaxinone in suppressing transcript level of SL biosynthesis genes and in alleviating Striga infestation under greenhouse conditions, and did not negatively impact mycorrhization. Taken together, MiZax are a promising tool for elucidating zaxinone biology and investigating rice development, and suitable candidates for combating Striga and increasing crop growth., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

As members of the plant microbiota, arbuscular mycorrhizal fungi (AMF) may be effective in enhancing plant resilience to drought, one of the major limiting factors threatening crop productivity. AMF host their own microbiota and previous data demonstrated that endobacteria thriving in Gigaspora margarita modulate fungal antioxidant responses. Here, we used the G. margarita - Candidatus Glomeribacter gigasporarum system to test whether the tripartite interaction between tomato, G. margarita and its endobacteria may improve plant resilience to combined water/nutrient stress. Tomato plants were inoculated with spores containing endobacteria (B+) or not (B-), and exposed to combined water/nutrient stress. Plants traits, AM colonization and expression of AM marker genes were measured. Results showed that mycorrhizal frequency was low and no growth effect was observed. Under control conditions, B+ inoculated plants were more responsive to the symbiosis, as they showed an up-regulation of three AM marker genes involved in phosphate and lipids metabolism compared with B- inoculated or not-inoculated plants. When combined stress was imposed, the difference between fungal strains was still evident for one marker gene. These results indicate that the fungal endobacteria finely modulate plant metabolism, even in the absence of growth response.

Loss-of-function alleles of MLO ( Mildew Resistance Locus O ) confer broad-spectrum resistance to foliar infections by powdery mildew pathogens. Like pathogens, microbes that establish mutually beneficial relationships with their plant hosts, trigger the induction of some defense responses. Initially, barley colonization by the root endophyte Serendipita indica (syn. Piriformospora indica ) is associated with enhanced defense gene expression and the formation of papillae at sites of hyphal penetration attempts. This phenotype is reminiscent of mlo -conditioned immunity in barley leaf tissue and raises the question whether MLO plays a regulatory role in the establishment of beneficial interactions. Here we show that S. indica colonization was significantly reduced in plants carrying mlo mutations compared to wild type controls. The reduction in fungal biomass was associated with the enhanced formation of papillae. Moreover, epidermal cells of S. indica -treated mlo plants displayed an early accumulation of iron in the epidermal layer suggesting increased basal defense activation in the barley mutant background. Correspondingly, the induction of host cell death during later colonization stages was impaired in mlo colonized plants, highlighting the importance of the early biotrophic growth phase for S. indica root colonization. In contrast, the arbuscular mycorrhizal fungus Funneliformis mosseae displayed a similar colonization morphology on mutant and wild type plants. However, the frequency of mycorrhization and number of arbuscules was higher in mlo- 5 mutants. These findings suggest that MLO differentially regulates root colonization by endophytic and AM fungi., (Copyright © 2020 Hilbert, Novero, Rovenich, Mari, Grimm, Bonfante and Zuccaro.)

Next-generation approaches have enabled researchers to deeply study the plant microbiota and to reveal how microbiota associated with plant roots has key effects on plant nutrition, disease resistance, and plant development. Although early "omics" experiments focused mainly on the species composition of microbial communities, new "meta-omics" approaches such as meta-transcriptomics provide hints about the functions of the microbes when interacting with their plant host. Here, we used an RNA-seq dataset previously generated for tomato ( Solanum lycopersicum ) plants growing on different native soils to test the hypothesis that host-targeted transcriptomics can detect the taxonomic and functional diversity of root microbiota. Even though the sequencing throughput for the microbial populations was limited, we were able to reconstruct the microbial communities and obtain an overview of their functional diversity. Comparisons of the host transcriptome and the meta-transcriptome suggested that the composition and the metabolic activities of the microbiota shape plant responses at the molecular level. Despite the limitations, mining available next-generation sequencing datasets can provide unexpected results and potential benefits for microbiota research., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Modifications in cell wall composition, which can be accompanied by changes in its structure, were already reported during plant interactions with other organisms, such as the mycorrhizal fungi. Arbuscular mycorrhizal (AM) fungi are among the most widespread soil organisms that colonize the roots of land plants, where they facilitate mineral nutrient uptake from the soil in exchange for plant-assimilated carbon. In AM symbiosis, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. In addition, to improve host nutrition and tolerance/resistance to environmental stresses, AM symbiosis was shown to modulate fruit features. In this study, Comprehensive Microarray Polymer Profiling (CoMMP) technique was used to verify the impact of the AM symbiosis on the tomato cell wall composition both at local (root) and systemic level (fruit). Multivariate data analyses were performed on the obtained datasets looking for the effects of fertilization, inoculation with AM fungi, and the fruit ripening stage. Results allowed for the discernment of cell wall component modifications that were correlated with mycorrhizal colonization, showing a different tomato response to AM colonization and high fertilization, both at the root and the systemic level., Competing Interests: The authors declare no conflict of interest.

Several studies have investigated soil microbial biodiversity, but understanding of the mechanisms underlying plant responses to soil microbiota remains in its infancy. Here, we focused on tomato (Solanum lycopersicum), testing the hypothesis that plants grown on native soils display different responses to soil microbiotas. Using transcriptomics, proteomics, and biochemistry, we describe the responses of two tomato genotypes (susceptible or resistant to Fusarium oxysporum f. sp. lycopersici) grown on an artificial growth substrate and two native soils (conducive and suppressive to Fusarium). Native soils affected tomato responses by modulating pathways involved in responses to oxidative stress, phenol biosynthesis, lignin deposition, and innate immunity, particularly in the suppressive soil. In tomato plants grown on steam-disinfected soils, total phenols and lignin decreased significantly. The inoculation of a mycorrhizal fungus partly rescued this response locally and systemically. Plants inoculated with the fungal pathogen showed reduced disease symptoms in the resistant genotype in both soils, but the susceptible genotype was partially protected from the pathogen only when grown on the suppressive soil. The 'state of alert' detected in tomatoes reveals novel mechanisms operating in plants in native soils and the soil microbiota appears to be one of the drivers of these plant responses., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Strigolactones (SLs), a novel class of plant hormones, are key regulator of plant architecture and mediator of biotic interactions in the rhizosphere. Root-released SLs initiate the establishment of arbuscular mycorrhizal (AM) symbiosis by inducing spore germination and hyphal branching in AM fungi (AMF). However, these compounds also trigger the germination of root parasitic weeds, paving the way for deleterious infestation. Availability of SLs is required for investigating of their functions and also for application in agriculture. However, natural SLs are difficult to synthesize due to their complex structure and cannot be isolated at large scale, as they are released at very low concentrations. Therefore, there is a need for synthetic SL analogs. Recently, we reported on the development of simple SL analogs, methyl phenlactonoates (MPs), which show high SL activity in plants. Here, we investigate the effect of MP1, MP3 and the widely used SL-analog GR24 on AMF spore germination and host root colonization. Our results show that MP1 and MP3 inhibit AMF spore germination, but promote the intra-radical root colonization, both more efficiently than GR24. These results indicate that field application of MP1 and MP3 does not have negative impact on mycorrhizal fungi. In conclusion, our data together with the previously reported simple synthesis, high activity in regulating plant architecture and inducing Striga seed germination, demonstrate the utility of MP1 and MP3 as for field application in combating root parasitic weeds by inducing germination in host's absence.

Besides improved mineral nutrition, plants colonised by arbuscular mycorrhizal (AM) fungi often display increased biomass and higher tolerance to biotic and abiotic stresses. Notwithstanding the global importance of wheat as an agricultural crop, its response to AM symbiosis has been poorly investigated. We focused on the role of an AM fungus on mineral nutrition of wheat, and on its potential protective effect against Xanthomonas translucens. To address these issues, phenotypical, molecular and metabolomic approaches were combined. Morphological observations highlighted that AM wheat plants displayed an increased biomass and grain yield, as well as a reduction in lesion area following pathogen infection. To elucidate the molecular mechanisms underlying the mycorrhizal phenotype, we investigated changes of transcripts and proteins in roots and leaves during the double (wheat-AM fungus) and tripartite (wheat-AM fungus-pathogen) interaction. Transcriptomic and proteomic profiling identified the main pathways involved in enhancing plant biomass, mineral nutrition and in promoting the bio-protective effect against the leaf pathogen. Mineral and amino acid contents in roots, leaves and seeds, and protein oxidation profiles in leaves, supported the omics data, providing new insight into the mechanisms exerted by AM symbiosis to confer stronger productivity and enhanced resistance to X. translucens in wheat.

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G. margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Arbuscular mycorrhizal fungi (AMF) occur in the rhizosphere and in plant tissues as obligate symbionts, having key roles in plant evolution and nutrition. AMF possess endobacteria, and genome sequencing of the endobacterium Candidatus Glomeribacter gigasporarum revealed a reduced genome and a dependence on the fungal host. To understand the effect of bacteria on fungal fitness, we used next-generation sequencing to analyse the transcriptional profile of Gigaspora margarita in the presence and in the absence of its endobacterium. Genomic data on AMF are limited; therefore, we first generated a gene catalogue for G. margarita. Transcriptome analysis revealed that the endobacterium has a stronger effect on the pre-symbiotic phase of the fungus. Coupling transcriptomics with cell biology and physiological approaches, we demonstrate that the bacterium increases the fungal sporulation success, raises the fungal bioenergetic capacity, increasing ATP production, and eliciting mechanisms to detoxify reactive oxygen species. By using TAT peptide to translocate the bioluminescent calcium reporter aequorin, we demonstrated that the line with endobacteria had a lower basal intracellular calcium concentration than the cured line. Lastly, the bacteria seem to enhance the fungal responsiveness to strigolactones, the plant molecules that AMF perceive as branching factors. Although the endobacterium exacts a nutritional cost on the AMF, endobacterial symbiosis improves the fungal ecological fitness by priming mitochondrial metabolic pathways and giving the AMF more tools to face environmental stresses. Thus, we hypothesise that, as described for the human microbiota, endobacteria may increase AMF innate immunity.

The objective of this study is to evaluate Lotus japonicus transcriptomic responses to arbuscular mycorrhizal (AM) germinated spore exudates (GSEs), responsible for activating nuclear Ca(2+) spiking in plant root epidermis. A microarray experiment was performed comparing gene expression in Lotus rootlets treated with GSE or water after 24 and 48 h. The transcriptional pattern of selected genes that resulted to be regulated in the array was further evaluated upon different treatments and timings. In particular, Lotus rootlets were treated with: GSE from the pathogenic fungus Colletotrichum trifolii; short chitin oligomers (COs; acknowledged AM fungal signals) and long COs (as activators of pathogenic responses). This experimental set up has revealed that AM GSE generates a strong transcriptomic response in Lotus roots with an extensive defense-related response after 24 h and a subsequent down-regulation after 48 h. A similar subset of defense-related genes resulted to be up-regulated also upon treatment with C. trifolii GSE, although with an opposite trend. Surprisingly, long COs activated both defense-like and symbiosis-related genes. Among the genes regulated in the microarray, promoter-GUS assay showed that LjMATE1 activates in epidermal cells and root hairs.

Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Background: Tomato (Solanum lycopersicum) establishes a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. The formation of the mycorrhizal association in the roots leads to plant-wide modulation of gene expression. To understand the systemic effect of the fungal symbiosis on the tomato fruit, we used RNA-Seq to perform global transcriptome profiling on Moneymaker tomato fruits at the turning ripening stage., Results: Fruits were collected at 55 days after flowering, from plants colonized with Funneliformis mosseae and from control plants, which were fertilized to avoid responses related to nutrient deficiency. Transcriptome analysis identified 712 genes that are differentially expressed in fruits from mycorrhizal and control plants. Gene Ontology (GO) enrichment analysis of these genes showed 81 overrepresented functional GO classes. Up-regulated GO classes include photosynthesis, stress response, transport, amino acid synthesis and carbohydrate metabolism functions, suggesting a general impact of fungal symbiosis on primary metabolisms and, particularly, on mineral nutrition. Down-regulated GO classes include cell wall, metabolism and ethylene response pathways. Quantitative RT-PCR validated the RNA-Seq results for 12 genes out of 14 when tested at three fruit ripening stages, mature green, breaker and turning. Quantification of fruit nutraceutical and mineral contents produced values consistent with the expression changes observed by RNA-Seq analysis., Conclusions: This RNA-Seq profiling produced a novel data set that explores the intersection of mycorrhization and fruit development. We found that the fruits of mycorrhizal plants show two transcriptomic "signatures": genes characteristic of a climacteric fleshy fruit, and genes characteristic of mycorrhizal status, like phosphate and sulphate transporters. Moreover, mycorrhizal plants under low nutrient conditions produce fruits with a nutrient content similar to those from non-mycorrhizal plants under high nutrient conditions, indicating that AM fungi can help replace exogenous fertilizer for fruit crops.

The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca(2+) spiking in the Medicago truncatula root epidermis. Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca(2+) spiking responses and LC-MS to characterize targeted molecules in GSEs. This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca(2+) spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca(2+) spiking in M. truncatula ROCs. We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Strigolactones (SLs) are newly identified hormones that regulate multiple aspects of plant development, infection by parasitic weeds, and mutualistic symbiosis in the roots. In this study, the role of SLs was studied for the first time in the model plant Lotus japonicus using transgenic lines silenced for carotenoid cleavage dioxygenase 7 (LjCCD7), the orthologue of Arabidopsis More Axillary Growth 3. Transgenic LjCCD7-silenced plants displayed reduced height due to shorter internodes, and more branched shoots and roots than the controls, and an increase in total plant biomass, while their root:shoot ratio remained unchanged. Moreover, these lines had longer primary roots, delayed senescence, and reduced flower/pod numbers from the third round of flower and pod setting onwards. Only a mild reduction in determinate nodule numbers and hardly any impact on the colonization by arbuscular mycorrhizal fungi were observed. The results show that the impairment of CCD7 activity in L. japonicus leads to a phenotype linked to SL functions, but with specific features possibly due to the peculiar developmental pattern of this plant species. It is believed that the data also link determinate nodulation, plant reproduction, and senescence to CCD7 function for the first time.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts dependent for completion of their life cycle on plant carbohydrates, which they trade for mineral nutrients. Plant colonization by AM fungi is therefore expected to induce profound changes in plant carbon metabolism. We have previously observed that on one hand starch accumulation increases in responses to pre-symbiotic fungal signals and on the other hand, it decreases in mycorrhizal Lotus japonicus roots (Gutjahr et al. in New Phytol 183:53-61, 2009). To examine the importance of starch metabolism for AM development, we took advantage of a novel series of Lotus japonicus mutants impaired either in starch degradation or in synthesis. Normal AM colonization in all mutants indicated that defects in starch metabolism do not affect AM development and that carbohydrates can be supplied to the AM fungus without a requirement for starch synthesis. Furthermore, our experiments allowed us to characterize root starch dynamics in detail and point to continued turnover of starch in the degradation mutants in the presence of mycorrhiza.

• The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. • Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

*Arbuscular mycorrhizal symbiosis develops in roots; extensive cellular reorganizations and specific metabolic changes occur, which are mirrored by local and systemic changes in the transcript profiles. *A TOM2 microarray (c. 12 000 probes) has been used to obtain an overview of the transcriptional changes that are triggered in Solanum lycopersicum roots and shoots, as a result of colonization by the arbuscular mycorrhizal fungus Glomus mosseae. The cell-type expression profile of a subset of genes was monitored, using laser microdissection, to identify possible plant determinants of arbuscule development,. *Microarrays revealed 362 up-regulated and 293 down-regulated genes in roots. Significant gene modulation was also observed in shoots: 85 up- and 337 down-regulated genes. The most responsive genes in both organs were ascribed to primary and secondary metabolism, defence and response to stimuli, cell organization and protein modification, and transcriptional regulation. Six genes, preferentially expressed in arbusculated cells, were identified. *A comparative analysis only showed a limited overlap with transcript profiles identified in mycorrhizal roots of Medicago truncatula, probably as a consequence of the largely nonoverlapping probe sets on the microarray tools used. The results suggest that auxin and abscisic acid metabolism are involved in arbuscule formation and/or functioning.

* Nutrient exchange is the key symbiotic feature of arbuscular mycorrhiza (AM). As evidence is accumulating that plants sense presymbiotic factors from AM fungi and prepare for colonization, we investigated whether modifications in plant sugar metabolism might be part of the precolonization program. * Inoculation of Lotus japonicus roots in a double Millipore sandwich with the AM fungus Gigaspora margarita prevented contact between the symbionts but allowed exchange of signal molecules. Starch content was used as a marker for root carbohydrate status. * Mycorrhizal colonization of L. japonicus roots led to a decrease in starch concentration. In roots inoculated in the double sandwich, the polysaccharide accumulated after 1 wk and persisted for at least 4 wk. The response was absent in the castor myc(-) mutant, sym4-2, while transcript levels of both CASTOR and POLLUX were slightly enhanced in the wild-type L. japonicus roots, suggesting a requirement of the corresponding proteins for the starch-accumulation response. Exudates obtained from fungal spores germinated in the absence of the plant also induced starch accumulation in wild-type L. japonicus roots. * We conclude that factors released from germinating AM fungal spores induce changes in the root carbon status, possibly by enhancing sugar import, which leads to starch accumulation when colonization is prevented.

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca(2+) indicator aequorin to detect intracellular Ca(2+) changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca(2+) were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca(2+)-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca(2+) transient were constitutively released in the medium, and the induced Ca(2+) signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca(2+) response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

The function of fungal volatiles in fungal-plant interactions is poorly understood. The aim here was to address this lack of knowledge, focusing on truffles, ectomycorrhizal fungi that are highly appreciated for their aroma. The effect of volatiles released by truffles was tested on Arabidopsis thaliana in a closed chamber bioassay. The volatiles produced by Tuber melanosporum, Tuber indicum and Tuber borchii fruiting bodies inhibited A. thaliana in terms of root length and cotyledon leaf size, and in some cases induced a bleaching of the seedlings, thus indicating toxicity. Ten synthetic volatiles were tested in a similar way. The strongest inhibitory effect was observed with C(8) molecules such as 1-octen-3-ol, an alcohol with a typical 'fungal smell'. Two of these C(8) compounds were further tested to investigate their mechanism of action. 1-Octen-3-ol and trans-2-octenal induced an oxidative burst (hydrogen peroxide, H(2)O(2)) in the A. thaliana leaves as well as a strong increase in the activities of three reactive oxygen species (ROS)-scavenging enzymes. These results demonstrate that fungal volatiles inhibit the development of A. thaliana and modify its oxidative metabolism. Even though limited to laboratory observations, these results indicate the presence of a hitherto unknown function of fungal volatiles as molecules that mediate fungal-plant interactions.

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation.

A full-length cDNA showing high similarity to previously described CuZn superoxide dismutases (SODs) was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG 34). The corresponding gene sequence, named GmarCuZnSOD, is composed of four exons. As revealed by heterologous complementation assays in a yeast mutant, GmarCuZnSOD encodes a functional polypeptide able to confer increased tolerance to oxidative stress. The GmarCuZnSOD RNA was differentially expressed during the fungal life cycle; highest transcript levels were found in fungal structures inside the roots as observed on two host plants, Lotus japonicus and Medicago truncatula. These structures also reacted positively to 3,3'-diaminobenzidine, used to localize H2O2 accumulation. This H2O2 is likely to be produced by CuZnSOD activity since treatment with a chelator of copper ions, generally used to inhibit CuZnSODs, strongly reduced the 3,3'-diaminobenzidine deposits. A slight induction of GmarCuZnSOD gene expression was also observed in germinated spores exposed to L. japonicus root exudates, although the response showed variation in independent samples. These results provide evidence of the occurrence, in an arbuscular mycorrhizal fungus, of a functional SOD gene that is modulated during the life cycle and may offer protection as a reactive oxygen species-inactivating system against localized host defense responses raised in arbuscule-containing cells.

• The LjSym4 mutation leads to Lotus japonicus plants that are defective in arbuscular mycorrhiza (AM) development. • Two alleles of LjSym4 with different phenotypic strength are compared here. The development of AM was assessed by considering five parameters related to fungal structures present in root segments from wild-type and mutant plants. The distribution of intercellular hyphae was determined using semithin sections from resin-embedded roots. Cellular interactions were investigated ultrastructurally, whereas cell wall components from the host plant were identified using immunogold labeling. • In roots of Ljsym4-1 mutant, fungal hyphae were mostly restricted to the intercellular spaces of the cortex, indicating a block to infection by mutant cortical cells, which resulted in a very low number of arbuscules. • This observation suggests the presence of an additional, genetically defined 'checkpoint' for mycorrhizal development, located at the wall of cortical cells. The LjSym4 gene is therefore required for infection of both epidermal and cortical cells by AM fungi.