Candida Vannini
email: candida.vannini@uninsubria.it

Guido Domingo

Valentina Fiorilli

Daniel Garçia Seco

Mara Novero

Milena Marsoni

Florence Wisniewski-Dye

Marcella Bracale

Lionel Moulin

Paola Bonfante

Daniel Garçia Seco

Florence Wisniewski-Dyé

Proteomic analysis reveals how pairing of a Mycorrhizal fungus with plant growth-promoting bacteria modulates growth and defense in wheat

Keywords: Funneliformis mosseae, Azospirillum brasilense, Paraburkholderia graminis, Xanthomonas translucens, proteome, pathogens, bi-and tripartite interaction, wheat, growth and defense response Funneliformis mosseae, Azospirillum brasilense, Paraburkholderia graminis, Xanthomonas translucens, proteome, pathogens, bi-and tripartite interaction, wheat, growth and defense response

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Like humans, animals, and fungi, plants live among a variety of microbial species, which together comprise the plant microbiota [START_REF] Schlaeppi | The plant microbiome at work[END_REF]. Plant root-associated microbes have received increasing attention, starting from their taxonomic description [START_REF] Bulgarelli | Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota[END_REF][START_REF] Lundberg | Defining the core Arabidopsis thaliana root microbiome[END_REF] to their role in plant health [START_REF] Müller | The Plant Microbiota: Systems-Level Insights and Perspectives[END_REF]. Data generated by multiple omics approaches demonstrate that the plant microbiota does not represent a random assembly of microbes living in the soil.

On the contrary, plant microbiota composition is determined by an active host plant-driven selection process, which depends on the plant genotype, environmental conditions, and microbial interactions [START_REF] Durán | Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival[END_REF][START_REF] Hacquard | Microbiota and Host Nutrition across Plant and Animal Kingdoms[END_REF][START_REF] Saad | Tailoring plant-associated microbial inoculants in agriculture: a roadmap for successful application[END_REF][START_REF] Thiergart | Symbiosis Genes Impact Microbial Interactions between Symbionts and Multikingdom Commensal Communities[END_REF][START_REF] Uroz | Plant Symbionts Are Engineers of the Plant-Associated Microbiome[END_REF]. The complexity of the microbial community structure parallels the many beneficial functions currently assigned to the plant microbiota: stimulation of plant growth through phytohormone production, improvement of the plant nutrient status through the increased uptake of nutrients such as inorganic phosphate (Pi) and nitrogen (N) and increased availability of nutrients such as iron, greater tolerance to abiotic stress (e.g., drought) and biotic stress, and increased activation of plant innate immunity [START_REF] Hacquard | Interplay Between Innate Immunity and the Plant Microbiota[END_REF]. Many of these benefits have been traditionally associated with the so-called plant growthpromoting bacteria (PGPB), as well as with the root symbionts, such as arbuscular mycorrhizal (AM) fungi and N-fixing bacteria [START_REF] Lugtenberg | Fungal endophytes for sustainable crop production[END_REF]. Comparison of the data generated by cultureindependent approaches with the knowledge obtained from the investigation of controlled binary interactions of bacterial and fungal isolates with their host plant has led to the creation of the so-called synthetic communities (SynComs) [START_REF] Herrera Paredes | Design of synthetic bacterial communities for predictable plant phenotypes[END_REF][START_REF] Tsolakidou | Rhizosphere-enriched microbes as a pool to design synthetic communities for reproducible beneficial outputs[END_REF]. Simultaneous inoculation of the host plant with several different beneficial microbes allows the investigation of plant responses under controlled and reproducible conditions. These new tools therefore form the basis of the so-called microbial revolution, defined as the microbe-driven increase in crop productivity, leading to higher sustainability [START_REF] Baez-Rogelio | Next generation of microbial inoculants for agriculture and bioremediation[END_REF].

Together with rice and corn, wheat is one of the most important crops worldwide [START_REF] Fernie | De Novo Domestication: An Alternative Route toward New Crops for the Future[END_REF]. Recently, many studies have been conducted on wheat-associated microbes, providing a detailed list of bacteria and fungi associated with wheat plants under natural conditions [START_REF] Kuźniar | Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. 'Hondia') and the first report of microbiota in Triticum spelta L. (cv. 'Rokosz')[END_REF][START_REF] Mahoney | Community Structure, Species Variation, and Potential Functions of Rhizosphere-Associated Bacteria of Different Winter Wheat[END_REF][START_REF] Naylor | Drought and host selection influence bacterial community dynamics in the grass root microbiome[END_REF][START_REF] Pagé | Nitrogen-and phosphorus-starved Triticum aestivum show distinct belowground microbiome profiles[END_REF] or describing the core microbiome of wheat [START_REF] Simonin | Influence of plant genotype and soil on the wheat rhizosphere microbiome: evidences for a core microbiome across eight African and European soils[END_REF], thus revealing the ecological rules that regulate microbial assembly [START_REF] Hassani | Interactions and Coadaptation in Plant Metaorganisms[END_REF]. Ecological studies suggest that higher soil microbial diversity results in a greater resilience of the plant population [START_REF] Van Der Heijden | Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity[END_REF]. However, the assumption that a mixture of beneficial microbes automatically provides greater plant protection is an oversimplification [START_REF] Rosier | A perspective on inter-kingdom signaling in plant-beneficial microbe interactions[END_REF]. In this context, the responses of wheat to its microbiota are still unknown.

In this study, to disentangle the inherent complexity of plant-microbiota interactions [START_REF] Vorholt | Establishing Causality: Opportunities of Synthetic Communities for Plant Microbiome Research[END_REF], we followed a reductionist approach, where we selected two PGPB species, Azospirillum brasilense and Paraburkholderia graminis, which are associated with wheat plants under natural conditions, as well as an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae. We hypothesized that targeted inoculation of wheat plants with the AMF and one of the two PGPB could provide an experimentally tractable system for evaluating the outcome of the interaction between beneficial microbes. Previously, we demonstrated that inoculation with F. mosseae improved plant growth and enhanced bioprotection in wheat [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. The current study aimed to investigate the long-term local and systemic effects of P. graminis or A. brasilense on the wheat proteome in non-mycorrhizal and mycorrhizal plants. We compared the proteomic changes in wheat triggered by co-inoculation of PGPB and F. mosseae with those elicited by single inoculations. We also investigated the bioprotective effects of bipartite (wheat-PGPB) and tripartite (wheat-PGPB-AMF) interactions on wheat plants against the leaf pathogen, Xanthomonas translucens. While A. brasilense drastically altered the bioprotective effect of the AMF, P. graminis did not affect AMF-induced pathogen resistance. Overall, proteomic changes revealed the molecular mechanisms underlying the tripartite interaction and showed that the beneficial effects of the AMF on plants are differentially modulated by the plant-associated PGPB.

MATERIALS AND METHODS

Bacterial strains, mycorrhizal fungus and wheat genotype

Two plant growth promoting bacteria (PGPB), Azospirillum brasilense Sp245 (obtained from UMR Ecologie Microbienne, Villeurbanne) and Paraburkolderia graminis C4D1M (type strain of the species, LMG collection, Ghent, Belgium), one wheat pathogen, Xanthomonas translucens CFBP2054 (obtained from CFBP collection), and one mycorrhizal fungus, Funneliformis mosseae (BEG.12, MycAgro Lab, France) were used in our experiments.

In detail, A. brasilense strain Sp245 was isolated from wheat roots and was shown to stimulate root development and increase plant dry mass [START_REF] Kapulnik | Changes in root morphology of wheat caused by Azospirillum inoculation[END_REF], while the strain C4D1M of P. graminis was isolated from senescent corn roots and found to positively interact with different species of wheat (L. Moulin, personal communication).

Gfp-tagged derivatives were also included for cytology analyses: A. brasilense Sp245 eGFP carrying the pMP2444 plasmid eGFP, GmR [START_REF] Wisniewski-Dyé | Azospirillum genomes reveal transition of bacteria from aquatic to terrestrial environments[END_REF] and P. graminis C4D1M eGFP, constructed by triparental mating (using a Tn7 eGFP construct described in [START_REF] Norris | Stable, Site-Specific Fluorescent Tagging Constructs Optimized for Burkholderia Species[END_REF] with a single insertion of the Tn7 upstream of the glmS gene). A.

brasilense was cultivated at 28°C on LBA medium (Luria Broth low salt, agar) and P. graminis in YMA medium (yeast extract, 3 g; mannitol, 10 g; KH2PO4, 0.5 g; MgSO4, 0.2 g; NaCl, 0.1 g; agar, 18 g; distilled water, 1 L; pH 6.8) and grown overnight in the same broth medium for inoculation. Strains were stored at -80°c in 20% glycerol. X. translucens CFBP 2054 was grown at 28 °C on Peptone sucrose agar (PSA) medium, retrieved from Petri dish with sterile water to reach OD 0.5 for leaf clipping and infiltration assays.

The Triticum aestivum cv Chinese Spring was used for all experiments (seeds obtained from Valeria Terzi, CREA, Italy).

Plant material and plant inoculations

The methodologies have already been described in previous articles on the wheat response to Xanthomonas (Garcia-Seco et al., 2017a) and to mycorrhizal fungi [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. Twelve combinations were studied: 1). Control plants (C), 2) A. brasilense-inoculated plants (Az), 3) P. graminis-inoculated plants (P), 4) F. mosseae-inoculated plants (M), 5) C+Xanthomonas translucens (X), 6) Az+X, 7) P+X, 8) M+X, 9) Az+M, 10) P+M, 11) Az+M+X, 12) P+M+X. An overview of the experiment is given in Figure S1.

Seeds were disinfected by immersing for 40 min in a sodium hypochlorite solution and washed with sodium thiosulfate (Hurek et al., 1994) and pregerminated. The seedlings were transferred to pots containing a mix of sterile quartz sand + either the F. mosseae carrier inoculum substrate (the substrate without the fungus) for control and PGPB conditions, or the F. mosseae inoculum (30% v/v) for mycorrhizal and mycorrhizal+PGPB conditions. PGPB were inoculated directly after seedling transfer to pots, with 1 ml per plant at OD 1 from an overnight broth culture washed once and diluted with water.

For each inoculated condition, 10 pots containing 1 plant were used for phenotyping of root and fresh weight at 50 dpi, 5 pots were used for proteomic, and 5 pots for leaf-clipping assays with Xanthomonas translucens.

All plants were maintained under glasshouse conditions under cycles of 12 h of light at 21 °C and 50% relative humidity (RH) and 12 h of dark at 21 °C and 50% RH, watered twice a week with water, and once with a modified Long-Ashton solution containing a low phosphorous concentration (32 μM Na2HPO4*12H2O).

Spikes weight were measured separately in the mature plants at the end of their natural cycle. The spikes were threshed and 1000-kernel weights were determined.

For Xanthomonas infections, two types of inoculation were performed. A phenotyping leaf-clipping assay with scissors soaked in a 0.5 OD X. translucens culture was performed at 46 dpi on a first set of plants for phenotyping the length of the symptoms at 4-day post-clipping (dpc; starting point of the lesions) and 26 dpc. A second set of plants dedicated to proteomic analyses was infiltrated at 49 dpi with a 0.5 OD X. translucens culture using a microneedle, as described in Garcia-Seco et al. (Garcia-Seco et al., 2017b), and sampled the following day.

Evaluation of wheat roots microbial colonization

The mycorrhizal and mycorrhizal+PGPB roots were stained with 0.1% cotton blue and the level of mycorrhizal colonization was assessed as previously described [START_REF] Trouvelot | Mesure du taux de mycorhization VA d'un systeme radiculaire. Recherche de methods d'estimation ayant une signification fonctionnelle[END_REF].

For Colony Forming Unit (CFU) counting from plant roots, root fragments were weighted then pulverized with a FastPrep TM in tubes containing a ceramic bead in 500 µL of sterile water, centrifuged at 1000 rpm for 30 s and drops of 20 µL of serial dilutions were plated on bacterial media and counted 24 h later.

Proteomic analysis and data processing

Plant samples (root and leaves) were sampled at 50 dpi and pulverized with liquid nitrogen. The used protocol for total protein extraction was based on SDS and phenol extraction [START_REF] Wu | Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis[END_REF]. Then, samples were digested and analysed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) as described previously (Garcia-Seco et al., 2017b). Mass spectrometer raw files were analysed by MaxQuant (version 1.6.2.3, default parameters) against UniProt T. aestivum (Version 2017-1, 150,716 entries), Uniprot Rhizophagus irregularis (Version 2015-10, 29,847 entries), Uniprot P. graminis (Version 2015-10, 6,732 entries) and Uniprot A. brasilense (Version 2015-10, 7,636 entries). On January 2019, the UniProt T. aestivum database has been updated. Therefore, we obtained the updated protein IDs by BLAST search of our dataset against the Uniprot T. aestivum 2019 database (143,020 entries). Unknown proteins were annotated by BLAST search against the Uniprot viridiplantae database (Version 2019-01, 6,913,939 entries), taking the first hit with a valid annotation.

All MS proteomic data have been deposited in the ProteomeXchange Consortium via PRIDE partner repository with the Username: reviewer04430@ebi.ac.uk and Password: 2vlyEEVZ.

MaxQuant output files were processed as described earlier [START_REF] Vannini | Proteasome-mediated remodeling of the proteome and phosphoproteome during kiwifruit pollen germination[END_REF]. Only proteins detected in at least two of the three biological replicates (75%) sharing the same treatment and tissue were considered.

To compare the differences among analytical groups we performed an ANOVA based multiple samples coupled with Tukey test using the R package LIMMA. Only proteins with false discovery rate (FDR) below 0.01 were considered Differentially Abundant Proteins (DAPs) within the various comparisons. In order to produce a reliable and robust dataset, all proteins which gave one nonzero and two zero outcomes (two-time imputation) in at least one of the samples in each comparison were considered unreliable and therefore eliminated.

In order to use bioinformatic tools available only for A. thaliana, a local BLAST of T. aestivum proteins against the TAIR10 database (version 2012-05-07) was performed.

The enrichment analysis was performed using the Gene Ontology Resource (http://geneontology.org), running PANTHER algorithm with A. thaliana as background and FDR < 0.05 or using the AgriGO Singular Enrichment Analysis (SEA) compare tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php?method=compare), with A.

thaliana TAIR10_2017 protein database as background, default parameters and a FDR threshold of 0.05 [START_REF] Du | agriGO: a GO analysis toolkit for the agricultural community[END_REF].

Amino acid analysis

For the amino acids (AAs) extraction, 0.1 g of lyophilized samples were resuspended in 10 mL of 0.1% (v/v) formic acid in water/methanol (50:50). 10 μL of 10 mM deuterated internal standards (L-Phenyl-d5-alanine and L-alanine 15 N Met), were added. Free AAs were quantified by Liquid Chromatography

Tandem Mass Spectrometry (LC-MS/MS) as described previously [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF].

Total glucose and nitrogen content

Soluble sugars were extracted as described by [START_REF] Shi | Differences in Sugar Accumulation and Mobilization between Sequential and Non-Sequential Senescence Wheat Cultivars under Natural and Drought Conditions[END_REF] with minor modifications. Briefly, 0.2 g of leaves material were boiled (80°C) in ethanol 80% for 30 min. After centrifugation (13000g x 5 min) the supernatant was recovered. The extraction was repeated twice and all supernatants were collected. Sucrose in solution was hydrolysed with HCl (2% of HCl concentrated V/V) for 5 min at 90°C. After acid neutralization by KOH (5% of 5N KOH V/V) total glucose was estimated by the dinitrosalicylic (DNS) method [START_REF] Miller | Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar[END_REF].

Wheat root N content was determined by CHNS elemental analyzer Thermo

Fisher Scientific following the manufacturer's specifications. About 2-3 mg of sample for each replicate were weighed and placed in a tin capsule containing 9.5 to 10.5 mg of vanadium pentoxide. The N2 product by sample combustion was quantitatively determined through a separation with a gas chromatograph (GC) followed by a quantification using a thermal conductivity detector. Three tests were prepared for each sample.

Statistics

Phenotyping data of plant weight, CFU and lesion length were analyzed, depending on normality of data, by ANOVA followed by Tukey post-hoc test or Kruskal-Wallis test followed by Mann-Whitney pairwise comparisons, in R Environment (rstatix) and figures produced by ggplot.

Data from quantification of amino acid, total glucose and nitrogen were subjected to statistical analysis by ANOVA and Tukey post-hoc test.

RESULTS

PGPB and AMF impact plant growth

Wheat plants were inoculated with A. brasilense Sp245, P. graminis C4D1M,

A. brasilense Sp245 plus F. mosseae, or P. graminis C4D1M plus F. mosseae (hereafter referred to as Az, P, AzM, and PM plants, respectively) and grown under controlled conditions (Figure S1). The root and shoot biomass of these plants was determined at 50 days post-inoculation (dpi) and compared with that of mock-inoculated control (C) and F. mosseae only-inoculated (M) plants. In isolation, A. brasilense Sp245 exerted a strong positive effect on the growth of roots and shoots, whereas P. graminis C4D1M did not induce statistically significant growth of these organs (Figure 1). Monitoring these plants until seed production revealed that P. graminis significantly increased the seed yield, doubling the spike weight (Figure S2).

To determine whether the positive impact of the two PGPB on plant growth was associated with an efficient colonization process, bacteria on the root surface were counted at different time points. A. brasilense exhibited the greatest colonizing potential, with the bacterial count remaining constant across different time points. Colonization by P. graminis decreased with time to 1 × 10 2 colony forming units (CFU) at 21 dpi (Figure S3). The success of AMF colonization was evaluated at 50 dpi by calculating the total length of colonized roots (F%) and total number of arbuscules (A%) in plants inoculated with AMF alone or AMF plus PGPB (A. brasilense or P. graminis).

Colonization by the AMF resulted in abundant arbuscules in all plants. No differences were detected in F% and A% among plants inoculated with AMF alone or together with PGPB, indicating that the presence or absence of PGPB does not affect mycorrhizal colonization (Figure S3).

The shoot weight of AzM and PM plants was significantly higher than that of Az and P plants (Figure 1B) but comparable with that of M plants, indicating that PGPB did not lead to any additional yield benefit compared with the mycorrhizal condition.

AMF alone or in combination with a PGPB triggers different responses to

X. translucens infection

Inoculated wheat plants were assessed for protective effect to X. translucens [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF] or in combination with P. graminis increased protection against X. translucens. Overall, this experiment showed that A. brasilense inoculation alone did not protect wheat plants against the pathogen, and rather undermined the positive effect exerted by F. mosseae.

leaf

Quantitative overview of proteomics analysis

We conducted proteomic analysis of the roots (R) of Az, P, M, AzM, and PM plants (hereafter referred to as RAz, RP, RM, RAzM, and RPM samples, respectively) as well as the leaves of these plants (hereafter referred to as LAz, LP, LM, LAzM, and LPM samples, respectively). We also performed proteomic analysis of the leaves of these plants following infection with X.

translucens (hereafter referred to as LAzX, LPX, LMX, LAzMX, and LPMX, respectively). Each treatment was analyzed in triplicate.

A total of 3,846 and 3,883 wheat proteins were identified and quantified in root and leaf samples, respectively. Samples were clustered by condition according to their protein expression patterns (Figure S4). Replicates within each analytical group clustered together, confirming the high reproducibility of biological replicates. Protein abundance was compared between samples, and differentially abundant proteins (DAPs) were identified using the following thresholds: false discovery rate (FDR) < 0.01 and log2fold-change (log2FC) > 0.5 (Tables S1-S5 and S7-S15).

Functional characterization of DAPs was conducted with Gene Ontology (GO) enrichment analysis to determine the main biological processes stimulated by microbial inoculations.

In root samples, approximately 7%, 0.6%, and 0.6% of all identified proteins were assigned to AMF, A. brasilense, and P. graminis proteomes, respectively, confirming the presence of all three root-associated microbes at harvest.

Wheat response to single inoculation: an overview

A large number of significant DAPs (P < 0.01) were identified; 639 DAPs (386 in leaves and 253 in roots) in the C vs. Az comparison, and 1,085 DAPs (424 in leaves and 661 in roots) in the C vs. P comparison (Tables S1-S4). In leaves, approximately 50% of the DAPs were common between the C vs. Az and C vs.

P comparisons (Figure S5A). By contrast, in roots, proteomic expression was highly specific, mirroring the stronger impact on the colonized niche; only 12% of the DAPs were common between the C vs. Az and C vs. P comparisons (Figure S5B).

To decipher the molecular mechanisms involved in A. brasilenseinduced plant growth promotion, we performed GO enrichment analysis of LAz vs LC, LP vs LC, Raz vs RC and RP vs RC (Figure 3). In leaf samples, "photosynthesis light harvesting" and "photosynthetic electron transport chain" were the two most enriched GO terms. These data were consistent with the higher total glucose content of LAz samples compared with LC, LP, and LM samples (Figure 4A). Our experiments, therefore, confirmed that A.

brasilense exhibits a greater ability to drive an increase in the glucose content of shoots than other beneficial microorganisms (F. mosseae and P. graminis).

Moreover, the LAz sample showed a higher abundance of sucrose transporter 2D (SUT2D) than the LC sample; SUT2D shows high similarity to rice SUT2 (OsSUT2), which is involved in sucrose mobilization to sink cells [START_REF] Eom | Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth[END_REF].

The "amino acid metabolism" GO term was highly enriched in enzymes involved in the synthesis of aspartate, proline, and branched-chain amino acids.

The increased content of amino acids both in root and leaf samples validated the proteomics data (Table S6). The presence of A. brasilense on wheat roots also increased the abundance of the phosphate transporter Pht1-10, which is induced by the AMF [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF] and enhances Pi uptake.

Interestingly, the abundance of Pht1-10 was lower in the RP sample vs RC (Figure S6). Moreover, Pht1-10 was among the four proteins whose abundance showed opposite trends between RAz and RP samples (Table S5). Plants assimilate and metabolize ammonium (NH4 + ) provided by diazotrophs, including Azospirillum spp. [START_REF] Carvalho | Nitrogen signalling in plant interactions with associative and endophytic diazotrophic bacteria[END_REF]. In LAz samples, we observed an increase in the abundance of ferredoxin-glutamate dehydrogenase, 2-oxoglutarate (2-OxG)/malate translocator, and isocitrate dehydrogenase (ICDH), and a decrease in the abundance of ferredoxin-nitrite reductase; both these trends are indicative of a higher NH4 + assimilation rate in the leaves of Az plants. The increase in photosynthesis could contribute to enhanced tolerance to NH4 + toxicity by increasing NH4 + assimilation [START_REF] Setién | High irradiance improves ammonium tolerance in wheat plants by increasing N assimilation[END_REF]. Higher concentrations of free amino acids and N in RAz samples support these proteomic results (Table S6, Figure 4B).

In roots and leaves, inoculation with A. brasilense stimulated the mitochondrial electron transport for ATP synthesis, even if the proteins involved were different (Tables S1 andS2).

Proteomic data showed that P. graminis inoculation also had a substantial impact on primary metabolism (glycolysis, tricarboxylic acid cycle, and aerobic respiration) in roots and leaves (Figures 3, Figure S6, Tables S3-S5).

In the LP sample, DAPs related to photosynthesis did not showed a clear pattern (Table S4).

Similar to Az plants, P. graminis-inoculated plants showed significantly higher concentrations of N and almost all amino acids than C plants, particularly in the roots (Figure 4B, Table S6). In P plants, the more efficient N uptake could be due to the increased abundance of the high-affinity nitrate transporter NAR2 and its activator NRT2. The abundance of the wheat ortholog of rice ammonium-inducible transporter 1-2 (OsAMT1-2) was also increased in RP sample (Figure S6, Table S5).

Overall, most of these proteomic changes affecting respiration, photosynthesis, N assimilation, and mineral nutrition mirror the differential growth response of wheat upon PGPB inoculation, as illustrated in Figure 1, with a significant systemic effect of only A. brasilense on plant growth.

AMF plays a dominant role in plant roots upon binary association with PGPB

We previously showed that F. mosseae elicits a significant proteomic change in wheat roots and leaves during colonization [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. Consistent with this observation, AzM and PM samples showed high numbers of DAPs;

RAzM vs. RC and RP vs. RC comparisons revealed 709 and 1055 DAPs, respectively (Tables S7 andS8), whereas LAzM vs. LC and LPM vs. LC comparisons revealed 504 and 808 DAPs, respectively (Tables S9 andS10, Figure S7).

Venn diagrams showed a specific contribution by F. mosseae in the roots of co-inoculated plants, mainly AzM plants (Figure S7B andS7D). These data are consistent with our previous proteomic data showing that F. mosseae has a stronger local but a weaker systemic impact on wheat [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. In roots, inoculation with F. mosseae alone or together with A. brasilense or P.

graminis led to an increased abundance of 196 proteins, several of which have been previously shown to increase in abundance during mycorrhization [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. In particular, we found increased levels of key enzymes involved in glycolysis and the pentose phosphate pathway (glyceraldehyde-3phosphate dehydrogenase, pyruvate kinase, and 6-phosphogluconate dehydrogenase), fatty acid biosynthesis and metabolism (plastid acetyl-CoA carboxylase and 3-oxoacyl-[acyl-carrier-protein] reductase, GDSL esterase/lipase), and mineral nutrition (OsAMT3;1 homolog). We also detected significant up-regulation of some proteins involved in plant defense, including one acidic endochitinase protein, cysteine-rich receptor-like protein kinase 25 (CRK25), and some Germin-like proteins (GLPs).

Venn diagrams also showed that most DAPs were exclusively expressed in AzM and PM plants. This trend was mainly detected in leaves (Figure S7A and S7C). In fact, GO enrichment analysis showed that oxidative phosphorylation, response to oxidative stress, photosynthesis, and response to abiotic stimulus were among the up-regulated biological processes in the LAzM vs. LPM comparison. Ribosome biogenesis, translation, and gene expression were down-regulated processes (Table S11, Figure S8).

Altogether, our results revealed that organ-specific proteomic changes in AMF-inoculated wheat plants [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF] are mostly maintained upon binary inoculation. This suggests that the AMF plays a dominant role in the root protein profile.

Defense proteins are induced locally and systemically during bipartite and tripartite interactions

Some rhizosphere-associated beneficial bacteria trigger a plant immunization phenomenon, called induced systemic resistance (ISR), thus priming the plant immune system (C. M. [START_REF] Pieterse | Induced systemic resistance by beneficial microbes[END_REF]. To verify whether A. brasilense and P. graminis possess the tools to elicit immune system priming in wheat, we first examined the enriched GO terms involved in plant-microbe interactions. Whereas the "response to wounding" was one of the up-regulated GO enriched categories in RAz samples, the DAPs enriched in the category "response to bacterium" showed a decrease, in LAz samples (Figure 3). In particular, we found reduced abundance of two chitinases, two pathogenesisrelated (PR) proteins, and one protein (A0A3B6BXY2) highly similar to the Arabidopsis heat stable 1 (HS1) protein, which exhibits antibacterial activity [START_REF] Park | Characterization of a heat-stable protein with antimicrobial activity from Arabidopsis thaliana[END_REF]. This decrease in the abundance of defense-related proteins in leaves could at least partly explain the susceptibility of Az plants to X.

translucens infection in leaf-clipping tests (Figure 2). The sucrose transporter SUT2D specifically induced by Azospirillum (Table S2) in wheat leaves could also play a role in the susceptibility of Az plants to pathogen infection.

Xanthomonas TAL effectors usually target SWEET family sugar transporters to sustain pathogen growth [START_REF] Verdier | Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae[END_REF].

In RP samples harvested at 50 dpi, one of the enriched GO terms was "defense response/incompatible interaction" with the induction of several defense proteins (Figure 3, Table S5). Among them, the A0A3B6DGK2 protein, which is similar to RPM1-interacting protein 4 (RIN4), a major regulator of plant defense that plays important roles in both pathogenassociated molecular pattern (PAMP)-triggered immunity (PTI) and effectortriggered immunity (ETI) [START_REF] Ray | Role of RIN4 in Regulating PAMP-Triggered Immunity and Effector-Triggered Immunity: Current Status and Future Perspectives[END_REF]. Additionally, lipoxygenase 1 (LOX1), 12-oxophytodienoate reductase 1 (OPR1), OPR3, and allene oxide cyclase 3 (AOC3) point to up-regulation of the biosynthesis of jasmonic acid (JA), a plant hormone that plays a key role in the biotic stress response and overall plant immunity (C. M. J. [START_REF] Pieterse | A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis[END_REF]. Moreover, RP samples showed an increased abundance of a protein (A0A3B6KT24) that is similar to the respiratory burst oxidase homolog protein D (RbohD) and is involved in the generation of reactive oxygen species (ROS) during incompatible plant-pathogen interactions [START_REF] Torres | /em> gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response[END_REF], a CERK homolog (A0A3B6RF20), and proteins required for lignin biosynthesis, which are activated in tomato plants associated with native microbiota (M. Chialva et al., 2018). Lastly, P. graminis-inoculated plants showed an increased abundance of some proteins involved in isoprene metabolism, suggesting the up-regulation of pathways involved in plant defense. These data support the hypothesis that similar to other PGPB (C. M. J. [START_REF] Pieterse | A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis[END_REF] and unlike A. brasilense, P. graminis elicits an immune response in the roots but does not elicit strong ISR at 50 dpi in the leaves against X. translucens infection (Figure 2).

To describe the systemic bioprotective effect of AMF and PGPB coinoculation in wheat, we performed LAzM vs. LAz and LPM vs. LP comparisons (Tables S12 andS13). Our analysis showed an increase in several DAPs, putatively involved in the biotic stress response, in both LAzM and LPM samples (Figure 5A). Among these DAPs, we found proteins either involved in JA biosynthesis, such as a phospholipase D, three LOXs, and an AOC, or induced by JA, such as a dirigent protein, PR4 (wheat protein) [START_REF] Desmond | Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum[END_REF], and OsMPK1 [START_REF] Singh | The rice MAPKK-MAPK interactome: the biological significance of MAPK components in hormone signal transduction[END_REF]. Moreover, we observed an increase in the abundance of other proteins involved in signaling of the plant immune response, such as the homolog of brassinosteroid (BR)-signaling kinase 1 (BSK1) [START_REF] Shi | BR-SIGNALING KINASE1 physically associates with FLAGELLIN SENSING2 and regulates plant innate immunity in Arabidopsis[END_REF] and a calcium-transporting ATPase, whose homolog (ACA8) is required for limiting the growth of virulent bacteria in Arabidopsis [START_REF] Frei Dit Frey | Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development[END_REF]. Another protein, whose expression was highly induced in LPM and LAzM samples, was manganese superoxide dismutase 1 (Mn-SOD1), which belongs to the polyphyletic family of enzymes and protect cells from reactive superoxide radical-induced damage, thus conferring increased stress tolerance (S. [START_REF] Wang | Mitochondrial Dysfunction Causes Oxidative Stress and Tapetal Apoptosis in Chemical Hybridization Reagent-Induced Male Sterility in Wheat[END_REF].

Overall, this analysis revealed some unexpected features: A. brasilense alone down-regulates plant defense (which is consistent with the observed disease susceptibility phenotype shown in Figure 2), while single inoculations of P. graminis and F. mosseae trigger a similar number of proteins involved in the plant immune response in an organ-dependent way. Co-inoculation of wheat plants with the AMF and A. brasilense or P. graminis elicits the plant immune response not only in LPM but also in LAzM samples, at least in the short term, suggesting that both microbial pairs (AMF-A. brasilense and AMF-P. graminis) induce a priming response at least at the proteome level (Figure 5A).

Microbial pairs modulate wheat response to X. translucens by inducing different proteomic changes

Leaf inoculation of M, Az, P, and C plants with X. translucens revealed that pathogen susceptibility detected in the leaves of LAzX plants was alleviated by the presence of mycorrhizal colonization at 4 dpc (based on the LAzMX vs.

LAzX comparison) (Figure 2A). To decipher the main proteins responsible for the reduction of symptoms at 4 dpc, we analyzed the DAPs identified in the LAzMX vs. LAzX and LAzMX vs. LMX comparisons (Tables S14 andS15).

The up-regulated proteins included those involved in JA biosynthesis and response. Several LOXs and two lipases including phospholipase A1-II and phospholipase D, which generate fatty acid substrates for JA biosynthesis [START_REF] Browse | Jasmonate Passes Muster: A Receptor and Targets for the Defense Hormone[END_REF], were highly induced in the LAzMX vs. LAzX and LAzMX vs. LMX comparisons [START_REF] Ishiguro | The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis[END_REF][START_REF] Lee | Lipases associated with plant defense against pathogens[END_REF][START_REF] Wang | Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis[END_REF][START_REF] Wasternack | Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in[END_REF]) (Figure 5A, Tables S14 andS15). In addition, two allene oxide synthase (AOS) enzymes, which catalyze the first step in the JA biosynthesis pathway, and two AOCs, which are committed for the second step in this pathway [START_REF] Schaller | Enzymes in jasmonate biosynthesis -structure, function, regulation[END_REF], were highly induced in LAzMX samples (Figure 5A) compared with LAzX and LMX samples. We also found that two JA-induced dirigentlike proteins, which act downstream of the JA biosynthesis pathway, were induced in LAzMX samples. This increase of proteins involved in JA biosynthesis was also observed in LPMX samples (Figure 5A).

Our results showed that A. brasilense-AMF and P. graminis-AMF interactions amplified JA signaling during pathogen attack. In addition, proteins involved in biotic stress, which were induced during A. brasilense-AMF and P. graminis-AMF interactions in LAzM and LPM samples, respectively (Figure 5A), were also recruited during X. translucens infection, as testified by their higher abundance in LAzMX and LPMX samples, respectively.

These proteomic data correlate with the reduced lesion length observed at the early time point (4 dpc) in AzMX plants with respect to AzX plants.

However, at 26 dpc, a significant reduction in lesion size resulted only in MX and PMX plants compared with CX plants (Figure 2B). These data suggest that when co-inoculated with A. brasilense, the bioprotective effect exerted of F.

mosseae is transient and probably related to prompt induction of the JA response.

Further analysis is needed to clarify the molecular changes during the later stage of pathogen attack under different conditions. However, the proteomic profile of LAzMX samples was very different from that of LPMX samples (799 DAPs; Table S16). Among the most abundant proteins identified in the LAzMX vs. LPMX comparison, we found proteins involved in the response to abiotic stimulus and oxidative stress, while those implicated in translation, ribosome biogenesis, gene expression were down-regulated. A similar pattern was already observed in the LAzM vs. LPM comparison (Figure S8).

Overall, these data highlight the intricate network of processes that regulate wheat-PGPB-AMF-pathogen interactions (as observed in LAzMX and LPMX samples). However, elicitation of defense priming in the proteome of LAzM and LPM samples does not necessarily lead to better performance once the plant is under pathogen attack.

DISCUSSION

Wheat, one of the earliest food crops to be domesticated, is currently the second most widely cultivated crop in the world and one of the most important grain sources for humans. Given the increasing relevance of plant microbiota, many researches have described wheat-associated microbiota by considering the effects in different organs as well as in grain production. The results of this study illustrate how proteomic changes in wheat plants depend on the inoculum composition (single or multiple microbes) and the organ under study, and lead to differential growth effects and pathogen resistance. All analyses revealed that the AMF was the crucial driver of plant growth and defense priming under our growth conditions (low P). However, the overall changes induced by the AMF-PGPB consortium can interfere with the final mycorrhizal-induced resistance (MIR) outcome (Figure 6).

Effect of beneficial microbes on wheat growth is organ-and microbial identity-dependent

In addition to their N-fixing ability, Azospirillum spp. exhibit a remarkable capacity to benefit a wide range of plant species by activating multiple mechanisms [START_REF] Fukami | Azospirillum: benefits that go far beyond biological nitrogen fixation[END_REF]; however, the available omics data are limited to the effects of A. brasilense inoculation on roots (Drogue et al., 2014;[START_REF] Spaepen | Phenotypical and molecular responses of Arabidopsis thaliana roots as a result of inoculation with the auxin-producing bacterium Azospirillum brasilense[END_REF]. In this study, we showed that the higher root and shoot biomass of plants colonized by A. brasilense is supported by the sustained activation of the main metabolic processes (respiration, photosynthesis, and N assimilation), while the roots act as a strong sink for nutrients, such as hexoses and amino acids.

These results are consistent with the findings of Zeffa and colleagues, who showed that A. brasilense promotes plant growth in maize by enhancing the plant photosynthetic potential or by increasing the N use efficiency [START_REF] Zeffa | Azospirillum brasilense promotes increases in growth and nitrogen use efficiency of maize genotypes[END_REF] On the other hand, P. graminis did not efficiently increase the root and shoot biomass of wheat plants but increased the spike biomass.

Wheat actively responds to P. graminis inoculation by eliciting many metabolic processes, which involve a higher number of DAPs compared with those induced by A. brasilense. Some of these processes were, however, common to the two bacterial species (e.g., processes involved in ROS scavenging) as well described for many other PGPB [START_REF] Fukami | Azospirillum: benefits that go far beyond biological nitrogen fixation[END_REF].

Wheat responds well to AMF, particularly F. mosseae [START_REF] Fiorilli | Omics approaches revealed how arbuscular mycorrhizal symbiosis enhances yield and resistance to leaf pathogen in wheat[END_REF]. The wheat-PGPB-AMF tripartite interaction led to intensive proteomic changes where nutrient transporters and many enzymes involved in primary and secondary metabolism, protein biosynthesis, and ROS homeostasis were elicited.

Overall, plant growth experiments, nutrient quantification, and proteomic analyses demonstrated that the AMF plays a leading role in tripartite interactions, particularly in the root, while PGBP (at least Azospirillum) affects systemic growth, as evident from the leaf proteome.

The bioprotective effect of the AMF is modulated by the nature of the coinoculated PGPB PGPB are considered essential components of the plant microbiota because of their ability to improve plant growth via multiple mechanisms, including plant health protection [START_REF] Berendsen | The rhizosphere microbiome and plant health[END_REF][START_REF] Lugtenberg | Fungal endophytes for sustainable crop production[END_REF]. Azospirillum is not a typical biocontrol agent, despite studies showing its ability to increase pathogen resistance in plants [START_REF] Bashan | Protection of Tomato Seedlings against Infection by Pseudomonas syringae pv. Tomato by Using the Plant Growth-Promoting Bacterium Azospirillum brasilense<[END_REF][START_REF] Kusajima | Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice[END_REF][START_REF] Tortora | Protection of strawberry plants (Fragaria ananassa Duch.) against anthracnose disease induced by Azospirillum brasilense[END_REF][START_REF] Yasuda | Effects of Colonization of a Bacterial Endophyte, Azospirillum sp. B510, on Disease Resistance in Rice[END_REF]. On the other hand, some

Paraburkholderia taxa, such as P. phytofirmans, induce resistance against a broad range of plant pathogens by inducing plant-mediated responses in aerial organs [START_REF] Miotto-Vilanova | Burkholderia phytofirmans PsJN Confers Grapevine Resistance against Botrytis cinerea via a Direct Antimicrobial Effect Combined with a Better Resource Mobilization[END_REF]. Proteomic analysis of wheat plants inoculated with a single microbe showed that proteins involved in plant defense were down-regulated in LAz samples. Moreover, according to the "pathogen starvation" model, which links plant resistance with soluble sugars [START_REF] Bezrutczyk | Sugar flux and signaling in plant-microbe interactions[END_REF], the high sugar and amino acid contents of LAz leaves coupled with an enhanced abundance of sugar transporters could guarantee a nutrientrich niche for the pathogen. Under these conditions, the plant could not activate any defense mechanisms, notwithstanding a light improvement in the presence of the AMF at 4 dpc.

P. graminis induced diverse proteomic changes in roots characterized by an increase in the abundance of proteins involved in microbe-associated molecular pattern (MAMP) perception, PTI and ETI regulation (RIN4), ROS production and detoxification, lignin biosynthesis, and isoprene metabolism.

These findings suggest that P. graminis elicits an immunomodulatory response; however, this does not lead to ISR.

The protein profiles clearly indicate the capacity of mycorrhizal plants, associated with PGPB, to increase the number of defense-related proteins in leaves in the absence of the pathogen, and an augmented capacity to express these proteins upon pathogen infection (Figure 5A). The up-regulation of JA biosynthesis proteins was a key finding because this hormone is considered the first regulator of the plant immune response [START_REF] Hickman | Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network[END_REF]; C. M. J. [START_REF] Pieterse | A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis[END_REF]. Several studies reported that AM symbiosis protects plants against pathogens, suggesting that JA defense mechanisms play a key role in MIR [START_REF] Jung | Mycorrhizainduced resistance and priming of plant defenses[END_REF].

AMF are a crucial component of the plant microbiota [START_REF] Bonfante | The mycobiota: fungi take their place between plants and bacteria[END_REF] and the first inducers of plant immunity. A previous study showed that co-inoculation of wheat with an AMF and Pseudomonas spp.

(PGPB) leads to synergistic effects, priming the host immunity through chitosan-induced callose deposition [START_REF] Pérez-De-Luque | The interactive effects of arbuscular mycorrhiza and plant growthpromoting rhizobacteria synergistically enhance host plant defences against pathogens[END_REF]. A comparable result has been described in tomato plants grown in native soil containing multiple bacteria and AMF; MAMPs released by various microbes enhance the plant immunity, thus activating PTI markers. When challenged by pathogenic Fusarium spp., the tomato plants were strongly protected because of the activation of specific antifungal proteins [START_REF] Chialva | Not only priming: Soil microbiota may protect tomato from root pathogens[END_REF].

According to a previously proposed hypothesis [START_REF] Cameron | Mycorrhiza-induced resistance: more than the sum of its parts?[END_REF], JA could act as a long distance signaling molecule that in mycorrhizal wheat, and also in the presence of both PGPB species, activates the systemic priming of plant defense. However, in our system, lesion length was reduced only at the early time point (4 dpc) in AzMX plants in comparison with AzX plants. At 26 dpc, a significant reduction in lesion length was observed only in MX and PMX plants in comparison with CX plants (Figure 2B). We speculate that additional determinants induced by the AMF-PGPB interactions interfere with cellular processes, leading to MIR. Proteomic data showed that in LAzMX samples, the abundance of proteins involved in ribosome biogenesis and gene expression decreased compared with their abundance in LMX and LBMX samples (Figure 5B). Ribosomal genes are highly responsive to stress and signaling molecules, indicating that the encoded proteins play roles in stress amelioration, besides house-keeping. The instantaneous up-regulation of ribosomal genes in response to stress might function as an prompt defense response [START_REF] Moin | Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation[END_REF]. In addition, a reduction in HMGA subfamily transcription factor and H2B and H1 histones could affect the transcription of defense-related genes [START_REF] Isaac | The non-host disease resistance response in peas; alterations in phosphorylation and ubiquitination of HMG A and histones H2A/H2B[END_REF]. Finally, a reduction in the abundance of two proteins involved in stomata regulation, H1.3 and ubiquitin-specific protease 24 [START_REF] Rutowicz | A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis[END_REF][START_REF] Zhao | Ubiquitin-specific protease 24 negatively regulates abscisic acid signalling in Arabidopsis thaliana[END_REF], and some proteins involved in cuticular wax production, could promote leaf pathogen invasion in AzMX plants.

CONCLUSION

Plant-associated microbiota hold great promise for the development of sustainable crop systems, and this can be guaranteed by the use of SynComs [START_REF] Kong | Paving the Way From the Lab to the Field: Using Synthetic Microbial Consortia to Produce High-Quality Crops[END_REF]. However, results obtained from on-field microbiota census and those obtained using reductionist approaches, mostly through laboratory-based experiments, have not yet been fully integrated [START_REF] Fitzpatrick | The Plant Microbiome: From Ecology to Reductionism and Beyond[END_REF]. Our results suggest that beneficial microbes have different impacts on plants, at least in wheat, and the plant growth-promoting effects of beneficial microbes are not always accompanied by enhanced pathogen resistance, as shown by A. brasilense inoculation (Figure 6). On the other hand, a bacterium that does not show strong growth-promoting effect, such as P. graminis, may be more effective against pathogen attack, if associated with an AMF (Figure 6). Our data highlight the crucial role of AM fungi, which are often absent in SynComs, as well as the potential contrasting effects of different AMF-PGPB consortia on plant defense. In a wider context, these findings suggest that SynCom efficiency should be validated by checking the outcome of the interaction under different conditions (microbe-microbe interactions; nutritional status, plant life cycle and biotic stress) before their exploitation for crop growth.

Figure S1 .

 S1 Figure S1. Overview of the experimental set up.

Figure S2 .

 S2 Figure S2. Spike fresh weight.

Figure S3 .

 S3 Figure S3. Evaluation of microbial root colonization.

Figure S4 .

 S4 Figure S4. Hierarchical clustering analysis of protein intensities.

Figure S5 .

 S5 Figure S5. Overlap of differentially abundant proteins (DAPs) between Az vs.

Figure S6 .

 S6 Figure S6. Heat map of DAPs belonging to "carbohydrate metabolism",

Figure S7 .

 S7 Figure S7. Venn diagrams of DAPs identified by comparing the proteome of

Figure S8 .

 S8 Figure S8. Schematic representation of AgriGO SEA COMPARE function of

Figure 1 .

 1 Figure 1. Effect of arbuscular mycorrhizal (AM) symbiosis on the biomass of

Figure 2 .

 2 Figure 2. Phenotypic evaluation of disease symptoms caused by the bacterial

Figure 3 .

 3 Figure 3. Gene Ontology (GO) enrichment analysis of DAPs identified by

Figure 4 .

 4 Figure 4. Total glucose and nitrogen (N) contents of wheat leaves and roots,

Figure 5 .Figure S5 .

 5S5 Figure 5. Heat map of the main DAPs involved in plant defense (A) and in cell

Figure S6 .

 S6 Figure S6. Heat map of DAPs belonging to "carbohydrate metabolism",

Figure S7 .

 S7 Figure S7. Venn diagrams of differentially abundant proteins (DAPs) found in

Figure S8 .

 S8 Figure S8. Schematic representation of AgriGO SEA COMPARE function of

  infection by leaf-clipping plants with the pathogen at 46 dpi and recording

	At 26 dpc, a significant reduction in symptoms was observed only in MX
	and PMX plants when compared with CX plants, whereas lesions were

leaf symptoms at 4 and 26 days post-leaf clipping (dpc). Pathogen-infected plants were identified as AzX, PX, MX, AzMX, and PMX and positive control plants as CX. Disease symptoms were evident in CX plants at 4 and 26 dpc (Figure 2). Lesion length in MX plants was significantly less than that in CX plants both at 4 and 26 dpc, consistent with our previous results (Fiorilli et al., 2018). Lesion length appeared extended in AzX and CX plants at both time points. AzMX plants showed reduced symptoms in comparison with AzX plants at 4 dpc but showed extended lesions compared with CX and AzX plants at 26 dpc. This result indicates that the F. mosseae-induced bioprotection in wheat is abrogated between 4 and 26 dpc in the presence of A. brasilense. significantly increased in AzX and AzMX plants compared with PX, MX, and PMX plants. These results indicate that inoculation with F. mosseae alone (

Table S5 .

 S5 Root-specific DAPs identified by comparing the root proteome of Az or P plants with that of C plants(RAz vs. RC and RP vs. RC comparisons,

	respectively).

Table S6 .

 S6 Amino acid content (ratio).

	Table S7. DAPs identified by comparing the root proteome of wheat plants co-
	inoculated with the arbuscular mycorrhizal fungus (AMF), Funneliformis
	mosseae, and A. brasilense (AzM) with that of C plants (RAzM vs. RC
	comparison).

Table S8 .

 S8 DAPs identified by comparing the root proteome of wheat plants coinoculated with the AMF and P. graminis (PM) with that of C plants (RPM vs.

	RC comparison).
	Table S9. DAPs identified by comparing the leaf proteomes of AzM and C
	plants (LAzM vs. LC comparison).
	Table S10. DAPs identified by comparing the leaf proteomes of PM and C
	plants (LPM vs. LC comparison).

Table S11 .

 S11 DAPs identified by comparing the leaf proteomes of AzM and PM plants (LAzM vs. LPM comparison).

Table S12 .

 S12 DAPs identified by comparing the leaf proteomes of Az plants treated with or without the AMF (LAzM vs. LAz comparison).

Table S13 .

 S13 DAPs identified by comparing the leaf proteomes of P plants treated with or without the AMF (LPM vs. LP comparison).

Table S14 .

 S14 DAPs identified by comparing the leaf proteomes of Az plants infected with or without the leaf pathogen, Xanthomonas translucens (LAzMX vs. LAzX comparison).

	Table S15. DAPs identified by comparing the leaf proteomes of AzM plants
	and AMF only-inoculated (M) plants infected with X. translucens (LAzMX
	vs. LMX comparison).

Table S16 .

 S16 DAPs identified by comparing the leaf proteomes of AzMX plants and PM plants infected with X. translucens (LAzMX vs. LPMX comparison).

Acknowledgments V.F., D.G.S., P.B., L.M., and C.V designed the study; V.F., D.G.S., M.N., and D.G. carried out the majority of experiments; G.D., M.M., and C.V performed the bioinformatic analysis of proteomic data; C.V., V.F., P.B. L.M., F.W.-D., and M.B. interpreted the data and wrote the manuscript. ACKNOWLEDGMENTS This work was supported by the MIC-CERES ("Microbial eco-compatible strategies for improving wheat quality traits and rhizospheric soil sustainability") Project (FC Project ID 2013-1888; AF Project ID 1301-003) jointly supported by the Agropolis Foundation (through the "Investissements d'avenir" program, with the reference number ANR-10-LABX-0001-01) and Fondazione Cariplo. The authors thank the Functional Genomics Center Zurich (FGCZ) for providing highly valuable technical support.

This work was supported by the MIC-CERES ("Microbial eco-compatible strategies for improving wheat quality traits and rhizospheric soil sustainability") Project (FC Project ID 2013-1888; AF Project ID 1301-003) jointly supported by the Agropolis Foundation (through the "Investissements d'avenir" program, with the reference number ANR-10-LABX-0001-01) and Fondazione Cariplo.

Funding

DATA AVAILABILITY STATEMENT

Data available on request from the authors

AUTHOR CONTRIBUTIONS

V.F., D.G.S., P.B., L.M., and C.V designed the study; V.F., D.G.S., M.N., and D.G. carried out the majority of experiments; G.D., M.M., and C.V performed the bioinformatic analysis of proteomic data; C.V., V.F., P.B. L.M., F.W.-D., and M.B. interpreted the data and wrote the manuscript.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

SUPPORTING INFORMATION

Table S1. Differentially abundant proteins (DAPs) identified by comparative proteomic analysis of the roots (R) of Azospirillum brasilense-inoculated (Az) and mock-inoculated (control; C) wheat plants (RAz vs. RC comparison). Table S2. DAPs identified by comparative proteomic analysis of the leaves (L) of Az and C plants (LAz vs. LC comparison).

Table S3. DAPs identified by comparing the leaf proteome of Paraburkholderia graminis-inoculated (P) and C plants (RP vs. RC comparison).

Table S4. DAPs identified by comparing the leaf proteome of P and C plants (LP vs. LC comparison).