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Uncovering the molecular dialogue between the arbuscular mycorrhizal fungi and the non-host plant.

Periodic Reporting for period 1 - AraMyco (Uncovering the molecular dialogue between the arbuscular mycorrhizal fungi and the non-host plant.)

Reporting period: 2015-05-01 to 2017-04-30

The arbuscular mycorrhizal (AM) symbiosis is one of the most widespread mutualisms on Earth. It is established between soil fungi from the phylum Glomeromycota and the roots of approximately 82% of the terrestrial plant species. In AM plants, an extensive network of fungal hyphae increases the plant’s exploratory capacity for water and mineral nutrients. In additions this AM symbiosis increase the plant tolerance against different abiotic and biotic stresses. In return, the host plant supplies the fungus with sugars. Even though widespread, not all plant species form mycorrhizal associations. These plants are denominated here as ‘non-host’ plants and include many important crops.

Better knowledge about how interact these AM fungi with host- and non-host plants is crucial to improve crops production quality and quantity.

The main goal of this study is to unravel the mechanisms that underlie this 'non-compatibility' between AM fungi and non-host plants. To achieve this objective we work with Arabidopsis thaliana, which is the model plant and belongs to the group of non-host plants.
To rise this main objective, some specific objectives were addressed:

To analyze the process involved during early or 'pre-symbiotic' stages of the interaction between AM fungi and non host plants.

To check the capacity of AM fungi to colonize non-host plants under different conditions.

To study differences and similarities on the transcriptomic profile in host and not host plants during the interaction with AM fungi.
A. thaliana seems to specifically recognize the AM fungi in initial stages of the interaction:
During the pre-symbiotic stage of an AM symbiosis both partners communicate through the exchange of diffusible molecules. Plant strigolactones have been identified as major contributors during host plant-AM fungi communication in pre-symbiotic stages. Upon perception of the AM fungal partner, strigolactone biosynthesis is induced in the host root. Interestingly, the strigolactone biosynthesis pathway is among the elements from the ‘symbiotic toolkit’ that are still present in the non-host A. thaliana. In order to study whether a strigolactone-based pre-symbiotic dialog is present in this AM incompatible interaction, we analyzed the activation of the A. thaliana strigolactone biosynthesis genes CCD7 and CCD8. We found that similar to AM-compatible interactions, the expression of both genes was induced in the non-host roots, after the perception of the AM fungus. These results suggest that the non-host plant A. thaliana might recognize actively the presence of the AM fungus Rhizophagus, increasing the expression of genes involved in the strigolactone biosynthesis pathway (CCD7 and CCD8). In order to further characterize the specificity of the R. irregularis-induced expression of CCD7 and CCD8, we tested the expression of these genes in roots in response to the pathogenic Fusarium oxysporum and the beneficial Trichoderma harzianum fungi. Our results show that neither the pathogen nor the beneficial fungus induced expression of CCD7 and CCD8. Our results suggest that the early induction of the strigolactone biosynthesis is still conserved in A. thaliana.

R. irregularis colonizes the non-host plant A. thaliana endophytically, inducing plant defense responses:
By using similar set-up as Veiga et al. (2103) we explored whether the non-host A. thaliana was able to establish a functional symbiosis when an active mycelium network was provided. We found that R. irregularis was able to colonize the root cortex of the non-host plant A. thaliana. However, arbuscules, which are the most characteristic structures in the AM symbiosis were not observed. Importantly, fungal colonization was only observed when R. irregularis was supported by a host plant, indicating the requirement of an active AM fungal network for A. thaliana colonization. These findings were further supported by the observation that transcripts of the constitutively expressed R. irregularis gene GintrRNA were expressed in A. thaliana roots grown in soil with an active mycorrhizal mycelium network. The absence of arbuscules in the cortex of A. thaliana indicates that the interaction between AM fungi and A. thaliana may not be functional. In order to verify this, we checked the expression of the fungal P and N transporter genes GintPT and GintAMT2, which are well-characterized markers for a functional AM symbiosis. The molecular analysis revealed that these genes were not up-regulated in R. irregularis when interacting with A. thaliana, further corroborating the absence of AM functionality. Collectively these results showing the ability of R. irregularis to colonize A. thaliana roots endophytically, and further demonstrating the absence of a functional AM symbiosis in this interaction.

In addition, we observed a strong growth reduction in shoot biomass of A. thaliana plants that were colonized by the AM fungus R. irregularis. This plant growth depression observed could be related to the non-compatible nature of the A. thaliana-R. irregularis interaction, which might induce costly defenses that in turn might compromise plant growth. To unravel the molecular mechanisms that underlie this plant growth reduction, a RNA-seq analysis was performed on A. thaliana roots after R. irregularis root colonization. To analyze the biological process triggered in the non-host plant A. thaliana after R. irregularis root colonization, a gene ontology (GO) terms analysis was performed. The analysis of the most significant GO terms over-represented in A. thaliana roots after R. irregularis colonization, showed the up-regulation of sulfur compounds related plant defense responses (i.e. glucosinolates and glycosinolates biosynthesis) and various categories associated to systemic acquired resistance. These results suggest that despite to the fact that the AM fungus R. irregularis can colonize A. thaliana roots in specific conditions, this colonization triggered plant defense responses in the non-host plant. This plant defense induction could explain the growth reduction observed in A. thaliana after R. irregularis root colonization.
The molecular and physiological mechanisms behind the inability of non-host plants to establish an AM symbiosis remain largely unknown. Our results indicate that at least some of the pre-symbiotic interactions between A. thaliana and AM fungi are still intact, as well that the AM fungus Rhizophagus irregularis, under certain conditions, was able to colonize A. thaliana root cortex. However this colonization does not trigger a functional symbiosis even showing some negative effects in A. thaliana colonized plants associated to a plant defense induction. This negative effect triggered by AM fungi root colonization in A. thaliana could be conserved in others non-host plants. Therefore we could use AM fungi as biological agents to control the growth of several weeds species, which belong to the non-host plant group, in agriculture fields.