Omyc analyzes reveal how plants respond to the symbiosis with the arbuscolar mycorrhizal fungi and symbiotic bacteria

Currently, there is an increasing body of evidence that the plant health depends on their tight associations with specialised soil microorganisms that have great effects on plant growth, protection and productivity. Among them, arbuscular mycorrhizal (AM) fungi (subphylum Glomeromycota, Schüßler et al., 2001) can establish a mutualistic association with most terrestrial plant species, including important agricultural and horticultural crops (Bonfante and Genre 2015). The AM symbiosis develops in roots, where the fungus colonizes the cortex supplying mineral nutrients to the plants in exchange of photosynthetic carbon compounds (Smith and Rweead, 2008). The exchanges between fungus and plant occur through specialized, branched and intracellular fungal structures called arbuscules (Bonfante and Anca 2009). Besides an improved mineral nutrition, plants colonized by AMF also receive other benefits, including an increased biomass, yield, and higher tolerance to biotic (pathogens) and abiotic (drought, salinity, heavy metals) stresses, leading to improved plant fitness (Gernns et al. 2001; Van der Heijden and Sanders 2002; Hildebrandt et al. 2007; Pozo and Azcón- Aguilar 2007; Aroca et al. 2008; Pozo et al., 2010; Lanfranco and Young 2012). Many plant roots also harbour a variety of soil beneficial bacteria that can benefit the plant by serving as plant-growth promoters. These bacteria, known as PGPR (plant-growth promoting rhizobacteria) can work as active rhizosphere components (Caballero-Mellado et al., 2007) or endophytic plant colonizers (Paungfoo-Lonhienne et al., 2014). The mechanisms that promote plant growth and development include: solubilization of minerals, nitrogen fixation, production of siderophores, plant growth regulators and organic acids, as well as protection by enzymes like chitinase, ACC-deaminase and glucanase (Berg, 2009; Glick et al., 2007; Hayat et al., 2010). These microorganisms can enhance biomass production and tolerance of the plants to several soil conditions as salinity, drought and heavy metals (Baharlouei et al., 2011). However, knowledge on the interaction of PGPR with many plants of agronomic interest is still scarce (Castanheira et al., 2015). Among plants colonized by AMF and PGPR is the bread wheat (Triticum aestivum), one of the most important food supply and the third-most widely grown crop worldwide, after rice and corn. Despite its economic and social importance, the effects of wheat symbiosis with the above microbes, considered individually or together, have never been investigated, in contrast to many other crop plants. Thanks to their benefits, AMF and PGPR can be used as natural biofertilizers and bioprotectors in integrated strategies for a sustainable and healthy wheat production. Furthermore, some AMF may contain endobacteria in their cytoplasm. These bacteria have a reduced genome, which lacks some crucial metabolic pathways and reveals dependence on the host for nutrients and energy (Ghignone et al. 2012). In exchange, they give many benefits to fungal host, sustaining its presymbiotic growth (Lumini et al., 2007; Salvioli et al., 2015), increasing the success of AMF sporulation and mechanisms for ROSdetoxification, and eliciting AMF innate immune responses (Salvioli et al., 2015). The beneficial effects of mycorrhizae in the rhizosphere are the result of synergistic interactions among all rhizosphere microbes, which are crucial for plant growth (Linderman, 1992). Thus, the relationship between AMF, their associated bacteria and plants provides a very interesting example of a metaorganism (Bosch and McFall-Ngai, 2011) and may be of great importance for sustainable agriculture. In view of the above observations, during this thesis work three aims were pursued. The first was the characterization of the molecular responses of wheat roots and leaves in presence of AMF alone and with a leaf pathogen to test whether AM fungi can be used as biofertilizer and bioprotector for enhancing plant growth and yield. We investigated the main pathways involved in enhancing plant biomass and mineral nutrition, and in promoting the bioprotective effect against a leaf pathogen. To address these issues, we combined phenotypic, metabolomic and molecular approaches, as detailed in Chapter 1. The second aim was to analyze how proteome of wheat, in both roots and leaves, changes in response to colonisation by AMF and PGPR, considering single or double inocula. The purpose was to use these rhizosphere microbes in integrated strategies for a sustainable agriculture to improve plant health and yield. We wanted to achieve information about proteins that play pivotal roles in the molecular interactions of wheat with AMF and PGPR. In particular, this study has been set to obtain proteomic data providing a comprehensive picture of the intricate and yet mostly unknown cross-talk between wheat and AMF/PGPR. Methods and results of this study are reported in Chapter 2. The third aim was to understand the effects of AMF endobacteria on both fungal and plant fitness. Thus, we analysed the proteomic profile of AMF spores with and without endobacteria, and after application of the synthetic strigolactone GR24 that, similarly to strigolactones produced by plant roots, is perceived by AM fungi, stimulating their energy metabolism and growth. The goal was to better explain with proteomics some morphological traits of the spore without bacteria. Moreover, we wanted to provide new insights into the molecular mechanisms mediating endosymbiosis and into how bacteria provide direct and/or indirect ecological benefits, not only for their fungal host, but also for the plant. A full account of this investigation is given in Chapter 3. The research activities carried out during this thesis can be divided in the following three distinct, but interconnected parts. In Chapter 1, we focused on the role of an AM fungus (Funneliformis mosseae) in the mineral nutrition of wheat, and on its potential protective effect against a leaf pathogen (Xanthomonas translucens). To address these issues, phenotypical, metabolomic and molecular approaches have been combined. Several studies have shown that both model and agricultural plants colonized by AMF often display an increased mineral nutrition and biomass, and higher tolerance to biotic and abiotic stresses. Despite wheat being one of the major global crops, its response to AM symbiosis has been poorly investigated so far. In this study, morphological observations indicated that AM wheat plants displayed a growth effect, in terms of biomass and grain yield, as well as a reduction of the lesions produced by the pathogen. To elucidate the molecular mechanisms underlying the mycorrhizal phenotype, we investigated the local and systemic changes of transcripts and proteins in roots and leaves during the bipartite (wheat-AM fungus) and tripartite (wheat-AM fungus-pathogen) interaction. Transcriptomic and proteomic profiling identified the main pathways (nutrient transport, primary metabolism, defence mechanisms, hormone regulation) involved in enhancing plant biomass, mineral nutrient content and in promoting the bio-protective effect against the leaf pathogen. Interestingly, the pathways were differentially regulated depending on the plant organ/microbe relationship. Mineral and amino-acid contents in roots, leaves and seeds, and protein oxidation profiles in leaves supported the omics data, providing new insightin the mechanisms exerted by AM symbiosis to confer stronger productivity and higher resistance to X. translucens in wheat. In Chapter 2, we studied the mechanisms behind PGPR (Burkholderia graminis) - wheat interactions and the synergic interaction between B. graminis and F. mosseae on plant, through a proteomic analysis of wheat roots and leaves. Thus, we investigated the proteome alterations triggered in wheat by the dual inoculation PGPR + AMF compared to the sum of the effects elicited by single inocula. The main pathways identified concerned regulation of metabolic process, phytohormones, mineral transport and stress responses. In plants inoculated with B. graminis, the regulation of proteins involved in auxin pathways and the increase of N uptake efficiency may explain the observed root growth increase upon PGPR inoculation. Moreover, bacteria promoted the increase of several proteins involved in abiotic stress, in particular salt stress, and may contribute to improvement of the plant performance under stress conditions. Dual inoculation further led to the activation of many growth and defense-related proteins in roots and at systemic level. This result indicates that the dual inoculation in wheat enhances the biofertilizer and bioprotective effects of PGPR B. graminis and AMF F. mossae when co-inoculated. Phenotypic results also revealed that dual inoculation stimulates the growth of both roots and leaves of wheat plants with respect to the control. Finally, in Chapter 3, we shed light on the interaction between AMF and their endosymbiont bacteria. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of its endobacterium Candidatus Glomeribacter gigasporarum indicated that endobacteria are able to enhance fungal bioenergetics capacity. iTRAQ quantitative proteomics was used to identify differentially expressed proteins in G. margarita germinating spores with its 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. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than 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.