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As obligate biotrophs that colonize the roots of most land plants, arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiota, since they are able to enhance host's nutrient acquisition. Some AMF possess endobacteria inside their cytoplasm, giving rise to a special type of endosymbiosis. Bacterial genome sequencing revealed a reduced genome and a dependence on the fungal host (Ghignone et al 2012, Torres et al..2015). Among them, Candidatus Glomeribacter gigasporarum (CaGg) has a positive influence on the physiology of its fungal host, the AMF Gigaspora margarita, rising its bioenergetic potential and enhancing its capability in detoxifying endogenous reactive oxygen species (Salvioli et al., 2016). Since a proteomic analysis revealed some changes in plant proteins which are markers of stress situations (Vannini et al, 2016), the purpose of this study was to verify whether the bacterial positive impact on the fungal physiology may be transmitted to the host plant. In order to test whether mycorrhizal plants were affected at transcriptional level depending on the endobacterial-fungal association, we performed an RNA-seq analysis of Lotus japonicus roots colonized by both the B+ (containing CaGg) and B- (without CaGg) G. margarita lines after 28 days. As expected, only a few genes (112) resulted to be differentially expressed: among them plant genes involved in hormones signaling, flavonoid biosynthesis and mineral exchange resulted to be sensitive to the presence or absence of the endobacterium inside the AMF. Interestingly, some genes involved in defense response to pathogens, such as PR10, were up-regulated in the B- colonized roots. The results suggest that endobacteria living inside the AM fungus may -directly or indirectly- influence the host plant responses.

Arbuscular mycorrhizal fungi (AMF) are crucial drivers of plant evolution: as obligate biotrophs, they have a key role in plant health. Some AMF possess endobacteria, whose genome sequencing revealed a reduced genome and a dependence on fungal host. We previously combined transcriptomics and cell biology to demonstrate that Candidatus Glomeribacter gigasporarum (CaGg) positively influences the physiology of its fungal host, Gigaspora margarita. We demonstrated how CaGg rises host's bioenergetic potential in terms of ATP production, enhancing its capability in detoxifying endogenous reactive oxygen species (Salvioli et al., 2016). Starting from these results we wondered whether such a positive impact of the bacterial presence on the fungal physiology may be transmitted to the host plant. With this aim, we performed an RNA-seq analysis of Lotus japonicus roots colonized by both the B+ (containing CaGg) and B- (without CaGg) G. margarita lines after 28 days. As expected, only a few genes (112) resulted to be differentially expressed: among them plant genes involved in hormones signaling, flavonoid biosynthesis and mineral exchange resulted to be sensitive to the presence or absence of the endobacterium inside the AMF. Interestingly, some genes involved in defense response to pathogens, such as PR10, were up-regulated in the B- colonized roots. The results suggest that the intracellular fungal microbiota may influence plant responses.

Root-associated microbiota play a major role in shaping plant physiology under diverse environmental conditions. Such an impact may have systemic outcomes, also influencing traits of agronomic relevance. In particular, arbuscular mycorrhizae (AM) improve mineral nutrition and fortify plants against biotic and abiotic stresses. We took advantage of next-generation sequencing to study interactions between Solanum lycopersicum (tomato) and its root-associated microbiota. Two agricultural soils (RO and AL) containing diverse microbiota and with different biotic and abiotic features were considered. The RO and AL soils were conducive and suppressive, respectively, to the pathogen Fusarium oxysporum f. sp. lycopersici (FOL). A steamed, peat-moss soil was used as a control. Two tomato genotypes (FOL-resistant and susceptible) were grown in microcosms containing the three soils without pathogen inoculation. After three months, roots were sampled, AM colonization was assessed, and the transcriptome was analyzed by RNAseq. Morphological observations indicated that AM structures were present in the roots from RO soil, while their presence was severely reduced in AL soil. RNAseq analysis indicates that the two soils, with their microbiota, shape the root transcriptome differently than the steamed soil. Moreover, the soil type was shown to cause a more relevant impact on gene expression than plant genotype, that is, AL soil activated metabolic pathways dealing with plant-defense irrespective of genetic background. However, comparing the profiles of the two genotypes, distinct sets of transcripts involved in plant-pathogen signaling emerged among the differentially expressed genes, suggesting that genotype is the second parameter that impacts the transcript profile interacting with the soil. Finally, a meta-transcriptome reconstruction confirmed the presence and activity of fungal communities strictly associated with the roots in both soils. Overall, our data sheds light on tomato responses to complex natural microbiota and suggest a strong interplay occurring between soil biotic and abiotic features, microbiota diversity, and genotype in tuning plant gene expression.

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 mosseaeand 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.

Phosphorus (P) is an essential plant nutrient and is limiting for plant growth in most natural ecosystems as well as in agricultural production throughout the world. In arbuscular mycorrhizal (AM) symbiosis, the fungus obtains carbon from the plant and transfers mineral nutrients, like phosphate (Pi), from the soil to the root cells. Mycorrhizal plants can acquire Pi directly from the soil through plant-specific Pi transporters (the direct uptake pathway) or through fungal uptake and transport systems (the mycorrhizal uptake pathway). The study of plant Pi transporters and of their functioning therefore represents a key point for plant physiology. In this work, after a morphological analysis of tomato mycorrhizal roots, we have used the laser microdissection technology to collect cortical, epidermal, and central cylinder cells with the aim of analyzing the expression of the five so far identified tomato Pi transporter genes in the different root tissues. Using reverse transcriptase PCR analysis, LePT1 and LePT2 transcripts were detected in the epidermal and cortical cells, while LePT3, LePT4, and LePT5 transcripts were confirmed to be exclusive of arbuscule-containing cells. Despite the presence of mRNA in all the analyzed cell populations, no expression of the known tomato Pi transporter genes was observed in the central cylinder cells, irrespective of the presence of the AM fungus.

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 C8 molecules such as 1-octen-3-ol, an alcohol with a typical `fungal smell'. Two of these C8 compounds were further tested to investigate their mechanism of action. 1-Octen-3-ol and trans-2-octenal induced an oxidative burst (hydrogen peroxide, H2O2) 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.

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 Ca2+ indicator aequorin to detect intracellular Ca2+ 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 Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ 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 (

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.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and demonstrated to be 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 the 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. Similarly to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were down-regulated in the cured line, as well as perturbation in lipid biosynthesis.