Summary: 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

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.

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H₂O₂) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H₂O₂ was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H₂O₂ 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 H₂O₂ 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 H₂O₂ 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 Ca²⁺-sensing aequorin system, increasing amounts of added H₂O₂ correlated with a higher cytosolic calcium ([Ca²⁺]_cyt) concentration. In MD and HW leaves, H₂O₂ also triggered the increase of [Ca²⁺]_cyt, but MD-elicited [Ca²⁺]_cyt increase was more pronounced when compared to HW leaves after addition of exogenous H₂O₂. 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.

• 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 (<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 Ca2+ 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.